Protein kinase stress-related proteins and methods of use in plants

ABSTRACT

A transgenic plant transformed by a Protein Kinase Stress-Related Protein (PKSRP) coding nucleic acid, wherein expression of the nucleic acid sequence in the plant results in the plants&#39;s increased growth under normal or stress conditions and/or increased tolerance to environmental stress as compared to a wild type variety of the plant. Also provided are agricultural products, including seeds, produced by the transgenic plants. Also provided are isolated PKSRPs, and isolated nucleic acid coding PKSRPs, and vectors and host cells containing the latter.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 10/768,863 filed Jan. 30, 2004, which is divisionalapplication of U.S. Nonprovisional patent application Ser. No.09/828,313 filed Apr. 6, 2001, which claims the priority benefit of U.S.Provisional Application Ser. No. 60/196,001 filed Apr. 7, 2000, all ofwhich are hereby incorporated in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to nucleic acid sequences encodingpolypeptides that are associated with increased growth and/or stressresistance in plants. In particular, this invention relates to nucleicacid sequences encoding polypeptides that confer upon a plant increasedgrowth under normal or stress conditions and/or increased toleranceunder abiotic stress conditions.

2. Background Art

Abiotic environmental stresses, such as drought stress, salinity stress,heat stress, and cold stress, are major limiting factors of plant growthand productivity. Crop losses and crop yield losses of major crops suchas rice, maize (corn), and wheat caused by these stresses represent asignificant economic and political factor and contribute to foodshortages in many underdeveloped countries.

Plants are typically exposed during their life cycle to conditions ofreduced environmental water content. Most plants have evolved strategiesto protect themselves against these conditions of desiccation. However,if the severity and duration of the drought conditions are too great,the effects on plant development, growth and yield of most crop plantsare profound. Furthermore, most of the crop plants are very susceptibleto higher salt concentrations in the soil. Continuous exposure todrought and high salt causes major alterations in the plant metabolism.These great changes in metabolism ultimately lead to cell death andconsequently yield losses.

Developing stress-tolerant plants is a strategy that has the potentialto solve or mediate at least some of these problems. However,traditional plant breeding strategies to develop new lines of plantsthat exhibit resistance (tolerance) to these types of stresses arerelatively slow and require specific resistant lines for crossing withthe desired line. Limited germplasm resources for stress tolerance andincompatibility in crosses between distantly related plant speciesrepresent significant problems encountered in conventional breeding.Additionally, the cellular processes leading to drought, cold, and salttolerance in model, drought- and/or salt-tolerant plants are complex innature and involve multiple mechanisms of cellular adaptation andnumerous metabolic pathways. This multi-component nature of stresstolerance has not only made breeding for tolerance largely unsuccessful,but has also limited the ability to genetically engineer stresstolerance plants using biotechnological methods.

Drought, cold as well as salt stresses have a common theme important forplant growth and that is water availability. Plants are exposed duringtheir entire life cycle to conditions of reduced environmental watercontent. Most plants have evolved strategies to protect themselvesagainst these conditions of desiccation. However, if the severity andduration of the drought conditions are too great, the effects on plantdevelopment, growth and yield of most crop plants are profound. Sincehigh salt content in some soils result in less available water for cellintake, its effect is similar to those observed under droughtconditions. Additionally, under freezing temperatures, plant cells loosewater as a result of ice formation that starts in the apoplast andwithdraws water from the symplast. Commonly, a plant's molecularresponse mechanisms to each of these stress conditions are common andprotein kinases play an essential role in these molecular mechanisms.

Plant biomass is yield for forage crops like alfalfa, silage corn, andhay. Many proxies for yield have been used in grain crops. Chief amongstthese are estimates of plant size. Plant size can be measured in manyways depending on species and developmental stage, but include totalplant dry weight, above-ground dry weight, above-ground fresh weight,leaf area, stem volume, plant height, rosette diameter, leaf length,root length, root mass, tiller number and leaf number. Many speciesmaintain a conservative ratio between the size of different parts of theplant at a given developmental stage. These allometric relationships areused to extrapolate from one of these measures of size to another (e.g.Tittonell et al 2005 Agric Ecosys & Environ 105: 213). Plant size at anearly developmental stage will typically correlate with plant size laterin development. A larger plant with a greater leaf area can typicallyabsorb more light and carbon dioxide than a smaller plant and thereforewill likely gain a greater weight during the same period (Fasoula &Tollenaar 2005 Maydica 50:39). This is in addition to the potentialcontinuation of the micro-environmental or genetic advantage that theplant had to achieve the larger size initially. There is a stronggenetic component to plant size and growth rate (e.g. ter Steege et al2005 Plant Physiology 139:1078), and so for a range of diverse genotypesplant size under one environmental condition is likely to correlate withsize under another (Hittalmani et al 2003 Theoretical Applied Genetics107:679). In this way a standard environment is used as a proxy for thediverse and dynamic environments encountered at different locations andtimes by crops in the field.

Harvest index, the ratio of seed yield to above-ground dry weight, isrelatively stable under many environmental conditions and so a robustcorrelation between plant size and grain yield can often be obtained(e.g. Rebetzke et al 2002 Crop Science 42:739). These processes areintrinsically linked because the majority of grain biomass is dependenton current or stored photosynthetic productivity by the leaves and stemof the plant (Gardener et al 1985 Physiology of Crop Plants. Iowa StateUniversity Press, pp 68-73) Therefore, selecting for plant size, even atearly stages of development, has been used as an indicator for futurepotential yield (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105:213). When testing for the impact of genetic differences on stresstolerance, the ability to standardize soil properties, temperature,water, and nutrient availability and light intensity is an intrinsicadvantage of greenhouse or plant growth chamber environments compared tothe field However, artificial limitations on yield due to poorpollination due to the absence of wind or insects, or insufficient spacefor mature root or canopy growth, can restrict the use of thesecontrolled environments for testing yield differences. Therefore,measurements of plant size in early development, under standardizedconditions in a growth chamber or greenhouse, are standard practices toprovide indication of potential genetic yield advantages.

There is a fundamental physiochemically-constrained trade-off, in allterrestrial photosynthetic organisms, between carbon dioxide (CO₂)absorption and water loss (Taiz and Zeiger, 1991, Plant Physiology,Benjamin/Cummings Publishing Co., p. 94). CO₂ needs to be in aqueoussolution for the action of CO₂ fixation enzymes such as Rubisco(Ribulose 1,5-bisphosphate Carboxylase/Oxygenase) and PEPC(Phosphoenolpyruvate carboxylase). As a wet cell surface is required forCO₂ diffusion, evaporation will inevitably occur when the humidity isbelow 100% (Taiz and Zeiger, 1991, p. 257). Plants have numerousphysiological mechanisms to reduce water loss (e.g. waxy cuticles,stomatal closure, leaf hairs, sunken stomatal pits). As these barriersdo not discriminate between water and CO₂ flux, these water conservationmeasures will also act to increase resistance to CO₂ uptake (Kramer,1983, Water Relations of Plants, Academic Press p. 305). PhotosyntheticCO₂ uptake is absolutely required for plant growth and biomassaccumulation in photoautotrophic plants.

Water Use Efficiency (WUE) is a parameter frequently used to estimatethe trade off between water consumption and CO₂ uptake/growth (Kramer,1983, Water Relations of Plants, Academic Press p. 405). WUE has beendefined and measured in multiple ways. One approach is to calculate theratio of whole plant dry weight, to the weight of water consumed by theplant throughout its life (Chu et al., 1992, Oecologia 89:580). Anothervariation is to use a shorter time interval when biomass accumulationand water use are measured (Mian et al., 1998, Crop Sci. 38:390).Another approach is to utilize measurements from restricted parts of theplant, for example, measuring only aerial growth and water use (Nienhuiset al 1994 Amer J Bot 81:943). WUE also has been defined as the ratio ofCO₂ uptake to water vapor loss from a leaf or portion of a leaf, oftenmeasured over a very short time period (e.g. seconds/minutes) (Kramer,1983, p. 406). The ratio of ¹³C/¹²C fixed in plant tissue, and measuredwith an isotope ratio mass-spectrometer, also has been used to estimateWUE in plants using C₃ photosynthesis (Martin et al., 1999, Crop Sci.1775).

An increase in WUE is informative about the relatively improvedefficiency of growth and water consumption, but this information takenalone does not indicate whether one of these two processes has changedor both have changed. In selecting traits for improving crops, anincrease in WUE due to a decrease in water use, without a change ingrowth would have particular merit in an irrigated agricultural systemwhere the water input costs were high. An increase in WUE driven mainlyby an increase in growth without a corresponding jump in water use wouldhave applicability to all agricultural systems. In many agriculturalsystems where water supply is not limiting, an increase in growth, evenif it came at the expense of an increased water use (i.e. no change inWUE), could also increase yield. Therefore new methods to increase bothWUE and biomass accumulation are required to improve agriculturalproductivity. As WUE integrates many physiological processes relating toprimary metabolism and water use, it is typically a highly polygenictrait with a large genotype by environment interaction (Richards et al.,2002, Crop Sci. 42:111). For these and other reasons, few attempts toselect for WUE changes in traditional breeding programs have beensuccessful.

Although some genes that are involved in stress responses and water useefficiency in plants have been characterized, the characterization andcloning of plant genes that confer stress tolerance and water useefficiency remains largely incomplete and fragmented. For example,certain studies have indicated that drought and salt stress in someplants may be due to additive gene effects, in contrast to otherresearch that indicates specific genes are transcriptionally activatedin vegetative tissue of plants under osmotic stress conditions. Althoughit is generally assumed that stress-induced proteins have a role intolerance, direct evidence is still lacking, and the functions of manystress-responsive genes are unknown.

There is a need, therefore, to identify additional genes expressed instress tolerant plants and plants that are efficient in water use thathave the capacity to confer stress tolerance and/or increased water useefficiency to the host plant and to other plant species. Newly generatedstress tolerant plants and plants with increased water use efficiencywill have many advantages, such as an increased range in which the cropplants can be cultivated, by for example, decreasing the waterrequirements of a plant species. Other desirable advantages includeincreased resistance to lodging, the bending of shoots or stems inresponse to wind, rain, pests, or disease.

Protein kinases represent a super family and the members of this familycatalyze the reversible transfer of a phosphate group of ATP to serine,threonine and tyrosine amino acid side chains on target proteins.Protein kinases are primary elements in signaling processes in plantsand have been reported to play crucial roles in perception andtransduction of signals that allow a cell (and the plant) to respond toenvironmental stimuli. In particular, receptor protein kinases (RPKs)represent one group of protein kinases that activate a complex array ofintracellular signaling pathways in response to the extracellularenvironment (Van der Gear et al., 1994 Annu. Rev. Cell Biol.10:251-337). RPKs are single-pass transmembrane proteins that contain anamino-terminal signal sequence, extracellular domains unique to eachreceptor, and a cytoplasmic kinase domain. Ligand binding induces homo-or hetero-dimerization of RPKs, and the resultant close proximity of thecytoplasmic domains results in kinase activation bytransphosphorylation. Although plants have many proteins similar toRPKs, no ligand has been identified for these receptor-like kinases(RLKs). The majority of plant RLKs that have been identified belong tothe family of Serine/Threonine (Ser/Thr) kinases, and most haveextracellular Leucine-rich repeats (Becraft, P W. 1998 Trends Plant Sci.3:384-388).

Another type of protein kinase is the Ca+-dependent protein kinase(CDPK). This type of kinase has a calmodulin-like domain at the COOHterminus, which allows response to Ca+ signals directly withoutcalmodulin being present. Currently, CDPKs are the most prevalentSer/Thr protein kinases found in higher plants. Although theirphysiological roles remain unclear, they are induced by cold, droughtand abscisic acid (ABA) (Knight et al., 1991 Nature 352:524; Schroeder,J I and Thuleau, P., 1991 Plant Cell 3:555; Bush, D. S., 1995 Annu. Rev.Plant Phys. Plant Mol. Biol. 46:95; Urao, T. et al., 1994 Mol. Gen.Genet. 244:331).

Another type of signaling mechanism involves members of the conservedSNF1 Serine/Threonine protein kinase family. These kinases playessential roles in eukaryotic glucose and stress signaling. PlantSNF1-like kinases participate in the control of key metabolic enzymes,including HMGR, nitrate reductase, sucrose synthase, and sucrosephosphate synthase (SPS). Genetic and biochemical data indicate thatsugar-dependent regulation of SNF1 kinases involves several othersensory and signaling components in yeast, plants and animals.

Additionally, members of the Mitogen-Activated Protein Kinase (MAPK)family have been implicated in the actions of numerous environmentalstresses in animals, yeasts and plants. It has been demonstrated thatboth MAPK-like kinase activity and mRNA levels of the components of MAPKcascades increase in response to environmental stress and plant hormonesignal transduction. MAP kinases are components of sequential kinasecascades, which are activated by phosphorylation of threonine andtyrosine residues by intermediate upstream MAP kinase kinases (MAPKKs).The MAPKKs are themselves activated by phosphorylation of serine andthreonine residues by upstream kinases (MAPKKKs). A number of MAP Kinasegenes have been reported in higher plants.

Casein kinase II (CK2) proteins represent a class of serine/theroninekinases that phosphorylate serine or theronine residues proximal toacidic amino acids. CK2 proteins have been demonstrated to phosphorylatea large number of physiological targets, and do not demonstrate thestrict specificity that other families of protein kinases exhibit forphosphorylation targets. The minimal consensus sequence forphosphorylation has been demonstrated to be Ser-Xaa-Xaa-Acidic, whereAcidic=Glu, Asp, pSer, or pTyr).

The functional CK2 enzyme has been demonstrated to consist of a tetramerof two catalytic α subunits and two regulatory β subunits. The CK2βregulatory subunit is almost completely conserved across organisms. TheCK2α catalytic subunit contains an N terminal core conserved kinasedomain and a more divergent C terminal domain of approximately 32 to 34amino acids. This divergent C terminal domain is recognized as thedistinguishing feature of distinct isoenzymes encoded by different geneswithin one organism (Lozeman, et al., 1990). It has been shown thatthese isoenzymes can partially complement knock out mice, but do notcompletely complement all phenotypes, indicating functional importanceof the isoenzymes and this C terminal divergent domain (Xu et al.,1999). This divergent C terminal domain may be of even more relevance asnew studies indicate that naturally occurring free CKα are commonlyfound in plants, perhaps with functions novel to the tetrameric complex(Filhol et al., 2004).

SUMMARY OF THE INVENTION

This invention fulfills in part the need to identify new, unique iontransporters capable of conferring stress tolerance and/or increasedgrowth under normal or stress conditions to plants upon modifyingexpression of genes. The present invention provides a transgenic plantcell transformed by a Protein Kinase Stress-Related Protein (PKSRP)coding nucleic acid, wherein expression of the nucleic acid sequence inthe plant cell results in increased growth under normal or stressconditions and/or increased tolerance to environmental stress ascompared to a wild type variety of the plant cell. Namely, describedherein are the protein kinases: 1) Ser/Thr Kinase and other type ofkinases (PK-6, PK-7, PK-8 and PK-9); 2) Calcium dependent proteinkinases (CDPK-1 and CDPK-2), 3) Casein Kinase homologs (CK-1, CK-2,CK-3, and CK2-1), and 4) MAP-Kinases (MPK-2, MPK-3, MPK-4 and MPK-5),all from Physcomitrella patens. Modifying expression of these PKSRPcoding nucleic acids in a plant results in the plant's increased growthunder normal or stress conditions and/or increased tolerance to anenvironmental stress.

Therefore, the present invention includes an isolated plant cellcomprising an ITSRP coding nucleic acid, wherein expression of thenucleic acid sequence in the plant cell results in increased growthunder normal or stress conditions and/or increased tolerance to anenvironmental stress as compared to a wild type variety of the plantcell. The invention provides in some embodiments that the PKSRP andcoding nucleic acid are that found in members of the genusPhyscomitrella. In another preferred embodiment, the nucleic acid andprotein are froma Physcomitrela patens. The invention provides that theenvironmental stress can be salinity, drought, nitrogen, temperature,metal, chemical, pathogenic and oxidative stresses, or combinationsthereof. In preferred embodiments, the environmental stress can bedrought, cold temperature or high salinity.

The invention further provides a seed produced by a transgenic planttransformed by a PKSRP coding nucleic acid, wherein the plant is truebreeding for increased growth under normal or stress conditions and/orincreased tolerance to an environmental stress as compared to a wildtype variety of the plant. The invention further provides a seedproduced by a transgenic plant expressing a PKSRP, wherein the plant istrue breeding for increased growth under normal or stress conditionsand/or increased tolerance to an environmental stress as compared to awild type variety of the plant.

The invention further provides an agricultural product produced by anyof the below-described transgenic plants, plant parts or seeds. Theinvention further provides an isolated PKSRP as described below. Theinvention further provides an isolated PKSRP coding nucleic acid,wherein the PKSRP coding nucleic acid codes for a PKSRP as describedbelow.

The invention further provides an isolated recombinant expression vectorcomprising a PKSRP coding nucleic acid as described below, whereinexpression of the vector in a host cell results in increased growthunder normal or stress conditions and/or increased tolerance toenvironmental stress as compared to a wild type variety of the hostcell. The invention further provides a host cell containing the vectorand a plant containing the host cell.

The invention further provides a method of producing a transgenic plantwith a PKSRP coding nucleic acid, wherein expression of the nucleic acidin the plant results in increased growth under normal or stressconditions and/or increased tolerance to environmental stress ascompared to a wild type variety of the plant comprising: (a)transforming a plant cell with an expression vector comprising a PKSRPcoding nucleic acid, and (b) generating from the plant cell a transgenicplant with an increased tolerance to environmental stress as compared toa wild type variety of the plant. In preferred embodiments, the PKSRPand PKSRP coding nucleic acid are as described below.

The present invention further provides a method of identifying a novelPKSRP, comprising (a) raising a specific antibody response to a PKSRP,or fragment thereof, as described below; (b) screening putative PKSRPmaterial with the antibody, wherein specific binding of the antibody tothe material indicates the presence of a potentially novel PKSRP; and(c) identifying from the bound material a novel PKSRP in comparison toknown PKSRP. Alternatively, hybridization with nucleic acid probes asdescribed below can be used to identify novel PKSRP nucleic acids.

The present invention also provides methods of modifying growth and/orstress tolerance of a plant comprising, modifying the expression of aPKSRP nucleic acid in the plant, wherein the PKSRP is as describedbelow. The invention provides that this method can be performed suchthat the plant growth and/or stress tolerance is either increased ordecreased. Preferably, the plant growth and/or stress tolerance isincreased in a plant via increasing expression of a PKSRP nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the correlation between the gene name and the SEQ ID NO inthe sequence listing.

FIG. 2 shows the results of a drought stress test with over-expressingPpPK-6 (SEQ ID NO: 14) transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 3 shows the results of a drought stress test with over-expressingPpPK-7 (SEQ ID NO:15) transgenic plants and wild-type Arabidopsis lines.The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 4 shows the results of a freezing stress test with over-expressingPpPK-7 (SEQ ID NO:15) transgenic plants and wild-type Arabidopsis lines.The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 5 shows the results of a drought stress test with over-expressingPpPK-9 (SEQ ID NO:17) transgenic plants and wild-type Arabidopsis lines.The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 6 shows the results of a freezing stress test with over-expressingPpPK-9 (SEQ ID NO:17) transgenic plants and wild-type Arabidopsis lines.The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 7 shows the results of a drought stress test with over-expressingPpCK-1 (SEQ ID NO:18) transgenic plants and wild-type Arabidopsis lines.The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 8 shows the results of a freezing stress test with over-expressingPpCK-1 (SEQ ID NO:18) transgenic plants and wild-type Arabidopsis lines.The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 9 shows the results of a drought stress test with over-expressingPpCK-2 (SEQ ID NO: 19) transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 10 shows the results of a drought stress test with over-expressingPpCK-3 (SEQ ID NO:20) transgenic plants and wild-type Arabidopsis lines.The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 11 shows the results of a drought stress test with over-expressingPpMPK-2 (SEQ ID NO:21) transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 12 shows the results of a freezing stress test with over-expressingPpMPK-2 (SEQ ID NO:21) transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 13 shows the results of a drought stress test with over-expressingPpMPK-3 (SEQ ID NO:22) transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 14 shows the results of a freezing stress test with over-expressingPpMPK-3 (SEQ ID NO:22) transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 15 shows the results of a drought stress test with over-expressingPpMPK-4 (SEQ ID NO:23) transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 16 shows the results of a drought stress test with over-expressingPpMPK-5 (SEQ ID NO:24) transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 17 shows the results of a drought stress test with over-expressingPpCPK-1 (SEQ ID NO:25) transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 18 shows the results of a drought stress test with over-expressingPpCPK-2 (SEQ ID NO:26) transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

FIG. 19 is a diagram illustrating the relative homology of the disclosedPpCK-4 (SEQ ID NO:130) and PpCK-3 (SEQ ID NO:33) amino acid sequenceswith other known CK2s. The diagram was generated using Align X of VectorNTI.

FIG. 20 shows the detailed alignment of the disclosed PpCK-4 (EST391,SEQ ID NO:130) and PpCK-3 (EST293, SEQ ID NO:33) amino acid sequenceswith other known CK2s. The alignment was generated using Align X ofVector NTI. Amino acids that are identical across all sequences areindicated with white text and black shading, amino acids that areconserved among sequences are indicated with black text and light greyshading, and amino acids that are similar over some or all of thesequences are indicated with white text and dark grey shading. Thealignment shows that the consensus sequence unique to PpCK2-1 (EST391,SEQ ID NO:130) and PpCK-3 (EST293, SEQ ID NO:33) is: YPXXXXXXXNR (SEQ IDNO:132), the consensus sequence of 80% of proteins is: VRXXEXSXXRXX (SEQID NO:133). Here, X represents any amino acid.

FIG. 21 is the homology table that shows the degree of amino acididentity of EST279 (PpPK-6, SEQ ID NO:27), EST451 (PpPK-7, SEQ IDNO:28), EST277 (PpPK-8, SEQ ID NO:29), EST357 (PpPK-9, SEQ ID NO:30),EST194 (PpCK-1, SEQ ID NO:31), EST263 (PpCK-2, SEQ ID NO:32), EST293(PpCK-3, SEQ ID NO:33), EST272 (PpMPK-2, SEQ ID NO:34), EST326 (PpMPK-3,SEQ ID NO:35), EST195 (PpMPK-4, SEQ ID NO:36), EST334 (PpMPK-5, SEQ IDNO:37), EST232 (PpCPK-1, SEQ ID NO:38), EST261 (PpCPK-2, SEQ ID NO:39),and EST391 (PpCK2-1, SEQ ID NO:130) (Pairwise Comparison was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum 62).

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. However, before the presentcompounds, compositions, and methods are disclosed and described, it isto be understood that this invention is not limited to specific nucleicacids, specific polypeptides, specific cell types, specific host cells,specific conditions, or specific methods, etc., as such may, of course,vary, and the numerous modifications and variations therein will beapparent to those skilled in the art. It is also to be understood thatthe terminology used herein is for the purpose of describing specificembodiments only and is not intended to be limiting. In particular, thedesignation of the amino acid sequences as protein “Protein KinaseStress-Related Proteins” (PKSRPs), in no way limits the functionality ofthose sequences.

The present invention provides a transgenic plant cell transformed by aPKSRP coding nucleic acid, wherein modifying expression of the nucleicacid sequence in the plant cell results in increased growth under normalor stress conditions and/or increased tolerance to environmental stressas compared to a wild type variety of the plant cell. The inventionfurther provides transgenic plant parts and transgenic plants containingthe plant cells described herein. Also provided is a plant seed producedby a transgenic plant transformed by a PKSRP coding nucleic acid,wherein the seed contains the PKSRP coding nucleic acid, and wherein theplant is true breeding for increased growth under normal or stressconditions and/or increased tolerance to environmental stress ascompared to a wild type variety of the plant. The invention furtherprovides a seed produced by a transgenic plant expressing a PKSRP,wherein the seed contains the PKSRP, and wherein the plant is truebreeding for increased growth under normal or stress conditions and/orincreased tolerance to environmental stress as compared to a wild typevariety of the plant. The invention also provides an agriculturalproduct produced by any of the below-described transgenic plants, plantparts and plant seeds.

As used herein, the term “variety” refers to a group of plants within aspecies that share constant characters that separate them from thetypical form and from other possible varieties within that species.While possessing at least one distinctive trait, a variety is alsocharacterized by some variation between individuals within the variety,based primarily on the Mendelian segregation of traits among the progenyof succeeding generations. A variety is considered “true breeding” for aparticular trait if it is genetically homozygous for that trait to theextent that, when the true-breeding variety is self-pollinated, asignificant amount of independent segregation of the trait among theprogeny is not observed. In the present invention, the trait arises fromthe transgenic expression of one or more DNA sequences introduced into aplant variety.

The present invention describes for the first time that thePhyscomitrella patens PKSRPs, PK-6, PK-7, PK-8, PK-9, CK-1, CK-2, CK-3,CK2-1, MPK-2, MPK-3, MPK-4, MPK-5, CPK-1 and CPK-2, are useful forincreasing a plant's growth under normal or stress conditions and/ortolerance to environmental stress. Accordingly, the present inventionprovides isolated PKSRPs selected from the group consisting of PK-6,PK-7, PK-8, PK-9, CK-1, CK-2, CK-3, CK2-1, MPK-2, MPK-3, MPK-4, MPK-5,CPK-l and CPK-2, and homologs thereof. In preferred embodiments, thePKSRP is selected from 1) Protein Kinase-6 (PK-6) protein as defined inSEQ ID NO:27; 2) Protein Kinase-7 (PK-7) protein as defined in SEQ IDNO:28; 3) Protein Kinase-8 (PK-8) protein as defined in SEQ ID NO:29; 4)Protein Kinase-9 (PK-9) protein as defined in SEQ ID NO:30; 5) CaseinKinase homologue (CK-1) protein as defined in SEQ ID NO:31; 6) CaseinKinase homologue-2 (CK-2) protein as defined in SEQ ID NO:32; 7) CaseinKinase homologue-3 (CK-3) protein as defined in SEQ ID NO:33; 8) MAPKinase-2 (MPK-2) protein as defined in SEQ ID NO:34; 9) MAP Kinase-3(MPK-3) protein as defined in SEQ ID NO:35; 10) MAP Kinase-4 (MPK-4)protein as defined in SEQ ID NO:36; 11) MAP Kinase-5 (MPK-5) protein asdefined in SEQ ID NO:37, 12) Calcium dependent protein kinase-1 (CPK-1)protein as defined in SEQ ID NO:38; 13) Calcium dependent proteinkinase-2 (CPK-2) protein as defined in SEQ ID NO:39; 14) Casein Kinasehomologus-4 (CK2-1) as defined in SEQ ID NO:130; and homologs andorthologs thereof. Homologs and orthologs of the amino acid sequencesare defined below.

The PKSRPs of the present invention are preferably produced byrecombinant DNA techniques. For example, a nucleic acid moleculeencoding the protein is cloned into an expression vector (as describedbelow), the expression vector is introduced into a host cell (asdescribed below) and the PKSRP is expressed in the host cell. The PKSRPcan then be isolated from the cells by an appropriate purificationscheme using standard protein purification techniques. Alternative torecombinant expression, a PKSRP polypeptide, or peptide can besynthesized chemically using standard peptide synthesis techniques.Moreover, native PKSRP can be isolated from cells (e.g., Physcomitrellapatens), for example using an anti-PKSRP antibody, which can be producedby standard techniques utilizing a PKSRP or fragment thereof.

The invention further provides an isolated PKSRP coding nucleic acid.The present invention includes PKSRP coding nucleic acids that encodePKSRPs as described herein. In preferred embodiments, the PKSRP codingnucleic acid is selected from 1) Protein Kinase-6 (PK-6) nucleic acid asdefined in SEQ ID NO:14; 2) Protein Kinase-7 (PK-7) nucleic acid asdefined in SEQ ID NO:15; 3) Protein Kinase-8 (PK-8) nucleic acid asdefined in SEQ ID NO:16; 4) Protein Kinase-9 (PK-9) nucleic acid asdefined in SEQ ID NO:17; 5) Casein Kinase homolog (CK-1) nucleic acid asdefined in SEQ ID NO:18; 6) Casein Kinase homolog-2 (CK-2) nucleic acidas defined in SEQ ID NO:19; 7) Casein Kinase homolog-3 (CK-3) nucleicacid as defined in SEQ ID NO:20; 8) MAP Kinase-2 (MPK-2) nucleic acid asdefined in SEQ ID NO:21; 9) MAP Kinase-3 (MPK-3) nucleic acid as definedin SEQ ID NO:22; 10) MAP Kinase-4 (MPK-4) nucleic acid as defined in SEQID NO:23; 11) MAP Kinase-5 (MPK-5) nucleic acid as defined in SEQ IDNO:24; 12) Calcium dependent protein kinase-1 (CPK-1) nucleic acid asdefined in SEQ ID NO:25; 13) Calcium dependent protein kinase-2 (CPK-2)nucleic acid as defined in SEQ ID NO:26; 14) Casein Kinase homologus-4(CK2-1) and/or as defined in SEQ ID NO:129 and homologs and orthologsthereof Homologs and orthologs of the nucleotide sequences are definedbelow. In one preferred embodiment, the nucleic acid and protein areisolated from the plant genus Physcomitrella. In another preferredembodiment, the nucleic acid and protein are from a Physcomitrellapatens (P. patens) plant.

As used herein, the term “environmental stress” refers to anysub-optimal growing condition and includes, but is not limited to,sub-optimal conditions associated with salinity, drought, temperature,nitrogen, metal, chemical, pathogenic and oxidative stresses, orcombinations thereof. In preferred embodiments, the environmental stresscan be salinity, drought, or temperature, or combinations thereof, andin particular, can be high salinity, low water content or lowtemperature. It is also to be understood that as used in thespecification and in the claims, “a” or “an” can mean one or more,depending upon the context in which it is used. Thus, for example,reference to “a cell” can mean that at least one cell can be utilized.

As also used herein, the terms “nucleic acid” and “nucleic acidmolecule” are intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. This term also encompassesuntranslated sequence located at both the 3′ and 5′ ends of the codingregion of the gene: at least about 1000 nucleotides of sequence upstreamfrom the 5′ end of the coding region and at least about 200 nucleotidesof sequence downstream from the 3′ end of the coding region of the gene.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one that is substantiallyseparated from other nucleic acid molecules, which are present in thenatural source of the nucleic acid. Preferably, an “isolated” nucleicacid is free of some of the sequences which naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated PKSRP nucleicacid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of nucleotide sequences which naturally flank thenucleic acid molecule in genomic DNA of the cell from which the nucleicacid is derived (e.g., a Physcomitrella patens cell). Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can be freefrom some of the other cellular material with which it is naturallyassociated, or culture medium when produced by recombinant techniques,or chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having a nucleotide sequence of SEQ ID NO:14, SEQ ID NO:15, SEQID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, or SEQ ID NO:129, or a portion thereof, can be isolated usingstandard molecular biology techniques and the sequence informationprovided herein. For example, a P. patens PKSRP cDNA can be isolatedfrom a P. patens library using all or portion of one of the sequences ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, orSEQ ID NO:129. Moreover, a nucleic acid molecule encompassing all or aportion of one of the sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:129 can be isolated bythe polymerase chain reaction using oligonucleotide primers designedbased upon this sequence. For example, mRNA can be isolated from plantcells (e.g., by the guanidinium-thiocyanate extraction procedure ofChirgwin et al., 1979 Biochemistry 18:5294-5299) and cDNA can beprepared using reverse transcriptase (e.g., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for polymerase chain reactionamplification can be designed based upon one of the nucleotide sequencesshown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26 or SEQ ID NO:129. A nucleic acid molecule of the inventioncan be amplified using cDNA or, alternatively, genomic DNA, as atemplate and appropriate oligonucleotide primers according to standardPCR amplification techniques. The nucleic acid molecule so amplified canbe cloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to a PKSRPnucleotide sequence can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises one of the nucleotide sequences shown in SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:129. These cDNAscomprise sequences encoding the PKSRPs (i.e., the “coding region”,indicated in Table 1), as well as 5′ untranslated sequences and 3′untranslated sequences. It is to be understood that SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26 or SEQ ID NO:129 comprise both coding regions and 5′and 3′ untranslated regions. Alternatively, the nucleic acid moleculesof the present invention can comprise only the coding region of any ofthe sequences in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ IDNO:129, or can contain whole genomic fragments isolated from genomicDNA. A coding region of these sequences is indicated as “ORF position.”The present invention also includes PKSRP coding nucleic acids thatencode PKSRPs as described herein. Preferred is a PKSRP coding nucleicacid that encodes a PKSRP selected from the group consisting of, PK-6(SEQ ID NO:27), PK-7 (SEQ ID NO;28), PK-8 (SEQ ID NO:29), PK-9 (SEQ IDNO:30), CK-l (SEQ ID NO:31), CK-2 (SEQ ID NO:32), CK-3 (SEQ ID NO:33),MPK-2 (SEQ ID NO:34), MPK-3 (SEQ ID NO:35), MPK4 (SEQ ID NO:36), MPK-5(SEQ ID NO:37), CPK-1 (SEQ ID NO:38), CPK-2 (SEQ ID NO:39) and CK2-1(SEQ ID NO:130), SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ IDNO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQID NO:142, and SEQ ID NO:143.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of one of the sequences in SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID) NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:129, for example, a fragmentwhich can be used as a probe or primer or a fragment encoding abiologically active portion of a PKSRP. The nucleotide sequencesdetermined from the cloning of the PKSRP genes from P. patens allow forthe generation of probes and primers designed for use in identifyingand/or cloning PKSRP homologs in other cell types and organisms, as wellas PKSRP homologs from other mosses and related species.

Portions of proteins encoded by the PKSRP nucleic acid molecules of theinvention are preferably biologically active portions of one of thePKSRPs described herein. As used herein, the term “biologically activeportion of” a PKSRP is intended to include a portion, e.g., adomain/motif, of a PKSRP that participates in a stress toleranceresponse in a plant, has an activity as set forth in Table 1, orparticipates in the transcription of a protein involved in a stresstolerance response in a plant. To determine whether a PKSRP, or abiologically active portion thereof, can participate in transcription ofa protein involved in a stress tolerance response in a plant, or whetherrepression of a PKSRP results in increased stress tolerance in a plant,a stress analysis of a plant comprising the PKSRP may be performed. Suchanalysis methods are well known to those skilled in the art, as detailedin Example 7. More specifically, nucleic acid fragments encodingbiologically active portions of a PKSRP can be prepared by isolating aportion of one of the sequences in SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141,SEQ ID NO:142, and SEQ ID NO:143, expressing the encoded portion of thePKSRP or peptide (e.g., by recombinant expression i l vitro) andassessing the activity of the encoded portion of the PKSRP or peptide.

Biologically active portions of a PKSRP are encompassed by the presentinvention and include peptides comprising amino acid sequences derivedfrom the amino acid sequence of a PKSRP, e.g., an amino acid sequence ofSEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:130, SEQ ID NO:134,SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ IDNO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143,or the amino acid sequence of a protein homologous to a PKSRP, whichinclude fewer amino acids than a full length PKSRP or the full lengthprotein which is homologous to a PKSRP, and exhibit at least oneactivity of a PKSRP. Typically, biologically active portions (e.g.,peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39,40, 50, 100 or more amino acids in length) comprise a domain or motifwith at least one activity of a PKSRP. Moreover, other biologicallyactive portions, in which other regions of the protein are deleted, canbe prepared by recombinant techniques and evaluated for one or more ofthe activities described herein. Preferably, the biologically activeportion of a PKSRP includes one or more selected domains/motifs orportions thereof having biological activity such as the conserved kinasedomain as are shown in FIG. 20. The ORF of PpCK-3 encodes the 333 aminoacid polypeptide shown in SEQ ID NO:33, including the casein kinase IIalpha subunit (CK2a) domain from amino acid position 34 to amino acidposition 319 of SEQ ID NO:33, the C terminal conserved functional domainfrom amino acid position 320 to amino acid position 330 of SEQ ID NO:33,and the C terminal conserved functional domain from amino acid position322 to amino acid position 333 of SEQ ID NO:33. The ORF of PpCK2-1encodes the 333 amino acid polypeptide shown in SEQ ID NO:130, includingthe casein kinase II alpha subunit (CK2α) domain from amino acidposition 34 to amino acid position 319 of SEQ ID NO:130, and the Cterminal conserved functional domain from amino acid position 320 toamino acid position 330 of SEQ ID NO:130, and the C terminal conservedfunctional domain from amino acid position 322 to amino acid position333 of SEQ ID NO:130.

In a preferred embodiment, the PKSRP comprises at least one of fiveconserved regions, wherein the first region commences with a methionineresidue at position 1, and has a serine residue at position 2, a lysineresidue at position 3, an alanine residue at position 4, a valineresidue at position 6, a tyrosine residue at position 7, an asparticacid residue at position 9, a valine residue at position 10, anasparagine residue at position 11, a valine residue at position 12, anarginine residue at position 14, a proline residue at position 15, atyrosine residue at position 18, a tryptophan residue at position 19, anaspartic acid residue at position 20, a tyrosine residue at position 21,a glutamic acid residue at position 22, a leucine residue at position24, a glutamine residue at position 27, a tryptophan residue at position28, a glycine residue at position 29, a glutamine residue at position31, an aspartic acid residue at position 32, an aspartic acid residue atposition 33, a tyrosine residue at position 34, a glutamic acid residueat position 35, a valine residue at position 36, a valine residue atposition 37, an arginine residue at position 38, a lysine residue atposition 39, a glycine residue at position 41, an arginine residue atposition 42, a glycine residue at position 43, a lysine residue atposition 44, a tyrosine residue at position 45, a serine residue atposition 46, a glutamic acid residue at position 47, a valine residue atposition 48, a phenylalanine residue at position 49, a glutamic acidresidue at position 50, a glycine residue at position 51, and anasparagine residue at position 53.

The second region is downstream from the first region, commences with alysine residue at position 1, and has an isoleucine residue at position2, a leucine residue at position 3, a lysine residue at position 4, aproline residue at position 5, a valine residue at position 6, a lysineresidue at position 7, a lysine residue at position 8, a lysine residueat position 9, an isoleucine residue at position 11, an arginine residueat position 13, a glutamic acid residue at position 14, an isoleucineresidue at position 15, a lysine residue at position 16, an isoleucineresidue at position 17, a leucine residue at position 18, a glutamineresidue at position 19, an asparagine residue at position 20, a leucineresidue at position 21, a cysteine residue at position 22, a glycineresidue at position 23, a glycine residue at position 24, a prolineresidue at position 25, an asparagine residue at position 26, anisoleucine residue at position 27, a valine residue at position 28, alysine residue at position 29, a leucine residue at position 30, anaspartic acid residue at position 32, a valine residue at position 34,an arginine residue at position 35, an aspartic acid residue at position36, a glutamine residue at position 37, a serine residue at position 39,a lysine residue at position 40, a threonine residue at position 41, aproline residue at position 42, a serine residue at position 43, aleucine residue at position 44, a phenylalanine residue at position 46,and a glutamic acid residue at position 47.

The third region is downstream from the second region, commences with anaspartic acid residue at position 1, and has a phenylalanine residue atposition 2, a lysine residue at position 3, a valine residue at position4, a leucine residue at position 5, a tyrosine residue at position 6, aproline residue at position 7, a threonine residue at position 8, aleucine residue at position 9, a threonine residue at position 10, anaspartic acid residue at position 11, an aspartic acid residue atposition 13, an isoleucine residue at position 14, an arginine residueat position 15, a tyrosine residue at position 16, a tyrosine residue atposition 17, an isoleucine residue at position 18, a glutamic acidresidue at position 20, a leucine residue at position 21, a leucineresidue at position 22, a lysine residue at position 23, an alanineresidue at position 24, a leucine residue at position 25, an asparticacid residue at position 26, a cysteine residue at position 28, ahistidine residue at position 29, a serine residue at position 30, aglutamine residue at position 31, a glycine residue at position 32, anisoleucine residue at position 33, a methionine residue at position 34,a histidine residue at position 35, an arginine residue at position 36,an aspartic acid residue at position 37, a valine residue at position38, a lysine residue at position 39, a proline residue at position 40, ahistidine residue at position 41, an asparagine residue at position 42,a valine residue at position 43, a methionine residue at position 44, anisoleucine residue at position 45, an aspartic acid residue at position46, a histidine residue at position 47, an arginine residue at position50, a lysine residue at position 51, a leucine residue at position 52,an arginine residue at position 53, a leucine residue at position 54, anisoleucine residue at position 55, an aspartic acid residue at position56, a tryptophan residue at position 57, a glycine residue at position58, a leucine residue at position 59, an alanine residue at position 60,a glutamic acid residue at position 61, a phenylalanine residue atposition 62, a tyrosine residue at position 63, a histidine residue atposition 64, a proline residue at position 65, a glycine residue atposition 66, a lysine residue at position 67, a glutamic acid residue atposition 68, a tyrosine residue at position 69, an asparagine residue atposition 70, a valine residue at position 71, an arginine residue atposition 72, a valine residue at position 73, an alanine residue atposition 74, a serine residue at position 75, an arginine at position76, a tyrosine residue at position 77, a phenylalanine residue atposition 78, a lysine residue at position 79, a glycine residue atposition 80, a proline residue at position 81, a glutamic acid residueat position 82, a leucine residue at position 83, a leucine residue atposition 84, a valine residue at position 85, an aspartic acid residueat position 86, a leucine residue at position 87, a glutamine residue atposition 88, an aspartic acid residue at position 89, a tyrosine residueat position 90, an aspartic acid residue at position 91, a tyrosineresidue at position 92, a leucine residue at position 94, an asparticacid residue at position 95, a methionine residue at position 96, atryptophan residue at position 97, a serine residue at position 98, aleucine residue at position 99, a glycine residue at position 100, acysteine residue at position I01, a methionine residue at position 102,a phenylalanine residue at position 103, an alanine residue at position104, a glycine residue at position 105, a methionine residue at position106, an isoleucine residue at position 107, a phenylalanine residue atposition 108, an arginine residue at position 109, a lysine residue atposition 110, a glutamic acid residue at position 111, a proline residueat position 112, a phenylalanine residue at position 113, aphenylalanine residue at position 114, a tyrosine residue at position115, a glycine residue at position 116, a histidine residue at position117, an aspartic acid residue at position 118, an asparagine residue atposition 119, an aspartic acid residue at position 121, a glutamineresidue at position 122, a leucine residue at position 123, a valineresidue at position 124, a lysine residue at position 125, an isoleucineresidue at position 126, a lysine residue at position 128, a valineresidue at position 129, a leucine residue at position 130, a glycineresidue at position 131, a threonine residue at position 132, and anaspartic acid residue at position 133.

The fourth region is downstream from the third region, commences with avaline residue at position 1, and has a glycine residue at position 2,an arginine residue at position 3, a histidine residue at position 4, aserine residue at position 5, an arginine residue at position 6, alysine residue at position 7, a proline residue at position 8, atryptophan residue at position 9, a serine residue at position 10, alysine residue at position 11, and a phenylalanine residue at position12. Finally, the fifth region is downstream from the fourth region,commences with a lysine residue at position 1, and has a leucine residueat position 2, a leucine residue at position 3, an arginine residue atposition 4, a tyrosine residue at position 5, an aspartic acid residueat position 6, a histidine residue at position 7, a glutamine residue atposition 8, an arginine residue at position 10, a leucine residue atposition 11, a threonine residue at position 12, an alanine residue atposition 13, a glutamic acid residue at position 15, an alanine residueat position 16, a methionine residue at position 17, an alanine residueat position 18, a histidine residue at position 19, a proline residue atposition 20, a tyrosine residue at position 21, and a phenylalanineresidue at position 22.

The invention also provides PKSRP chimeric or fusion proteins. As usedherein, a PKSRP “chimeric protein” or “fusion protein” comprises a PKSRPpolypeptide operatively linked to a non-PKSRP polypeptide. A PKSRPpolypeptide refers to a polypeptide having an amino acid sequencecorresponding to a PKSRP, whereas a non-PKSRP polypeptide refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the PKSRP, e.g., a protein thatis different from the PKSRP and is derived from the same or a differentorganism. Within the fusion protein, the term “operatively linked” isintended to indicate that the PKSRP polypeptide and the non-PKSRPpolypeptide are fused to each other so that both sequences fulfill theproposed function attributed to the sequence used. The non-PKSRPpolypeptide can be fused to the N-terminus or C-terminus of the PKSRPpolypeptide. For example, in one embodiment, the fusion protein is aGST-PKSRP fusion protein in which the PKSRP sequences are fused to theC-terminus of the GST sequences. Such fusion proteins can facilitate thepurification of recombinant PKSRPs. In another embodiment, the fusionprotein is a PKSRP containing a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of a PKSRP can be increased through use of aheterologous signal sequence.

Preferably, a PKSRP chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primers,which give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and re-amplified togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, Eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A PKSRPencoding nucleic acid can be cloned into such an expression vector suchthat the fusion moiety is linked in-frame to the PKSRP.

In addition to fragments and fusion proteins of the PKSRPs describedherein, the present invention includes homologs and analogs of naturallyoccurring PKSRPs and PKSRP encoding nucleic acids in a plant. “Homologs”are defined herein as two nucleic acids or proteins that have similar,or “homologous”, nucleotide or amino acid sequences, respectively.Homologs include allelic variants, orthologs, paralogs, agonists andantagonists of PKSRPs as defined hereafter. The term “homolog” furtherencompasses nucleic acid molecules that differ from one of thenucleotide sequences shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, orSEQ ID NO:129 (and portions thereof) due to degeneracy of the geneticcode and thus encode the same PKSRP as that encoded by the nucleotidesequences shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ IDNO:129. As used herein a “naturally occurring” PKSRP refers to a PKSRPamino acid sequence that occurs in nature. Preferably, a naturallyoccurring PKSRP comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:130,SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ IDNO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, andSEQ ID NO:143.

An agonist of the PKSRP can retain substantially the same, or a subset,of the biological activities of the PKSRP. An antagonist of the PKSRPcan inhibit one or more of the activities of the naturally occurringform of the PKSRP. For example, the PKSRP antagonist can competitivelybind to a downstream or upstream member of the cell membrane componentmetabolic cascade that includes the PKSRP, or bind to a PKSRP thatmediates transport of compounds across such membranes, therebypreventing translocation from taking place.

Nucleic acid molecules corresponding to natural allelic variants andanalogs, orthologs and paralogs of a PKSRP cDNA can be isolated based ontheir identity to the Physcomitrella patens PKSRP nucleic acidsdescribed herein using PKSRP cDNAs, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. In an alternative embodiment,homologs of the PKSRP can be identified by screening combinatoriallibraries of mutants, e.g., truncation mutants, of the PKSRP for PKSRPagonist or antagonist activity. In one embodiment, a variegated libraryof PKSRP variants is generated by combinatorial mutagenesis at thenucleic acid level and is encoded by a variegated gene library. Avariegated library of PKSRP variants can be produced by, for example,enzymatically ligating a mixture of synthetic oligonucleotides into genesequences such that a degenerate set of potential PKSRP sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofPKSRP sequences therein. There are a variety of methods that can be usedto produce libraries of potential PKSRP homologs from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene is then ligated into an appropriate expression vector.Use of a degenerate set of genes allows for the provision, in onemixture, of all of the sequences encoding the desired set of potentialPKSRP sequences. Methods for synthesizing degenerate oligonucleotidesare known in the art (see, e.g., Narang, S. A., 1983 Tetrahedron 39:3;Itakura et al., 1984 Annu. Rev. Biochem. 53:323; Itakura et al., 1984Science 198:1056; Ike et al., 1983 Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the PKSRP coding regions can beused to generate a variegated population of PKSRP fragments forscreening and subsequent selection of homologs of a PKSRP. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a PKSRP coding sequence witha nuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA, which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with SI nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the PKSRP.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of PKSRP homologs. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique that enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify PKSRP homologs (Arkin and Yourvan, 1992 PNAS 89:7811-7815;Delgrave et al., 1993 Protein Engineering 6(3):327-331). In anotherembodiment, cell based assays can be exploited to analyze a variegatedPKSRP library, using methods well known in the art. The presentinvention further provides a method of identifying a novel PKSRP,comprising (a) raising a specific antibody response to a PKSRP, or afragment thereof, as described herein; (b) screening putative PKSRPmaterial with the antibody, wherein specific binding of the antibody tothe material indicates the presence of a potentially novel PKSRP; and(c) analyzing the bound material in comparison to known PKSRP, todetermine its novelty.

To determine the percent homology of two amino acid sequences (e.g., oneof the sequences of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:130, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142,and SEQ ID NO:143 and a mutant form thereof), the sequences are alignedfor optimal comparison purposes (e.g., gaps can be introduced in thesequence of one protein or nucleic acid for optimal alignment with theother protein or nucleic acid). The amino acid residues at correspondingamino acid positions or nucleotide positions are then compared. When aposition in one sequence (e.g., one of the sequences of SEQ ID NO:27,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:130, SEQ ID NO:] 34, SEQ IDNO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143) is occupiedby the same amino acid residue as the corresponding position in theother sequence (e.g., a mutant form of the sequence selected from thepolypeptide of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:130,SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ IDNO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, andSEQ ID NO:143), then the molecules are homologous at that position(i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”). The same type ofcomparison can be made between two nucleic acid sequences.

The percent homology between the two sequences is a function of thenumber of identical positions shared by the sequences (i.e., % homology=numbers of identical positions/total numbers of positions x 100).Preferably, the amino acid sequences included in the present inventionare at least about 50-60%, preferably at least about 60-70%, and morepreferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, andmost preferably at least about 96%, 97%, 98%, 99% or more homologous toan entire amino acid sequence shown in SEQ ID NO:27, SEQ ID NO:28, SEQID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQiID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141,SEQ ID NO:142, or SEQ ID NO:143. In yet another embodiment, at leastabout 50-60%, preferably at least about 60-70%, and more preferably atleast about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and mostpreferably at least about 96%, 97%, 98%, 99% or more homologous to anentire amino acid sequence encoded by a nucleic acid sequence shown inSEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ r) NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:129. In otherembodiments, the preferable length of sequence comparison for proteinsis at least 15 amino acid residues, more preferably at least 25 aminoacid residues, and most preferably at least 35 amino acid residues. Inanother embodiment, the isolated amino acid homologs included in thepresent invention are at least about 50-60%, preferably at least about60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%,85-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%,99%, or more identical to the catalytic α subunits or the regulatory βsubunits of the disclosed amino acid sequences. As shown herein, theterm “protein kinase domain” refer to residues 31 to 319 of SEQ IDNO:130 or the corresponding region of SEQ ID NO:33 as showvn in FIG. 20.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleotide sequence which is at least about50-60%, preferably at least about 60-70%, more preferably at least about70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and even more preferably atleast about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotidesequence shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ IDNO:129, or a portion thereof The preferable length of sequencecomparison for nucleic acids is at least 75 nucleotides, more preferablyat least 100 nucleotides and most preferably the entire length of thecoding region.

It is also preferable that the homologous nucleic acid molecule of theinvention encodes a protein or portion thereof which includes an aminoacid sequence which is sufficiently homologous to an amino acid sequenceof SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, SEQ I) NO:39, SEQ ID NO:130, SEQ ID NO:134,SEQ I) NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ IDNO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143such that the protein or portion thereof maintains the same or a similarfunction as the amino acid sequence to which it is compared. Functionsof the PKSRP amino acid sequences of the present invention include theability to participate in a stress tolerance response in a plant, ormore particularly, to participate in the transcription of a proteininvolved in a stress tolerance response in a Physcomitrella patensplant. Examples of such activities are described in Table 1.

For the purposes of the invention, the percent sequence identity betweentwo nucleic acid or polypeptide sequences is determined using the VectorNTI 9.0 (PC) software package (Invitrogen, 1600 Faraday Ave., Carlsbad,Calif. 92008). A gap-opening penalty of 15 and a gap extension penaltyof 6.66 are used for determining the percent identity of two nucleicacids. A gap-opening penalty of 10 and a gap extension penalty of 0.1are used for determining the percent identity of two polypeptides. Allother parameters are set at the default settings. For purposes of amultiple alignment (Clustal W algorithm), the gap-opening penalty is 10,and the gap extension penalty is 0.05 with blosum62 matrix. It is to beunderstood that for the purposes of determining sequence identity whencomparing a DNA sequence to an RNA sequence, a thymidine nucleotide isequivalent to a uracil nucleotide.

In addition to the above-described methods, a determination of thepercent homology between two sequences can be accomplished using amathematical algorithm. A preferred, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990 Proc. Natl. Acad. Sci. USA90:5873-5877). Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul, et al. (1990 J. Mol. Biol. 215:403410).

BLAST nucleic acid searches can be performed with the NBLAST program,score=100, wordlength=12 to obtain nucleic acid sequences homologous tothe PKSRP nucleic acid molecules of the invention. Additionally, BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to PKSRPs of thepresent invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (1997Nucleic Acids Res. 25:3389-3402). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. Another preferred non-limiting exampleof a mathematical algorithm utilized for the comparison of sequences isthe algorithm of Myers and Miller (CABIOS 1989). Such an algorithm isincorporated into the ALIGN program (version 2.0) that is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12 and a gap penalty of 4 can be used toobtain amino acid sequences homologous to the PKSRPs of the presentinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al. (1997 NucleicAcids Res. 25:3389-3402). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. Another preferred non-limiting exampleof a mathematical algorithm utilized for the comparison of sequences isthe algorithm of Myers and Miller (CABIOS 1989). Such an algorithm isincorporated into the ALIGN program (version 2.0) that is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12 and a gap penalty of 4 can be used.

Finally, homology between nucleic acid sequences can also be determinedusing hybridization techniques known to those of skill in the art.Accordingly, an isolated nucleic acid molecule of the inventioncomprises a nucleotide sequence which hybridizes, e.g., hybridizes understringent conditions, to one of the nucleotide sequences shown in SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:129, or a portionthereof More particularly, an isolated nucleic acid molecule of theinvention is at least 15 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising anucleotide sequence of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ IDNO:129. In other embodiments, the nucleic acid is at least 30, 50, 100,250 or more nucleotides in length.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% homologous to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 65%, more preferably at leastabout 70%, and even more preferably at least about 75% or morehomologous to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, 6.3.1-6.3.6, John Wiley& Sons, N.Y. (1989). A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6X sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions toa sequence of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:129corresponds to a naturally occurring nucleic acid molecule. As usedherein, a “naturally occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein). In one embodiment, the nucleic acidencodes a naturally occurring Physcomitrella patens PKSRP.

Using the above-described methods, and others known to those of skill inthe art, one of ordinary skill in the art can isolate homologs of thePKSRPs comprising amino acid sequences shown in SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:135, SEQ IDNO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQID NO:141, SEQ ID NO:142, and SEQ ID NO:143. One subset of thesehomologs are allelic variants. As used herein, the term “allelicvariant” refers to a nucleotide sequence containing polymorphisms thatlead to changes in the amino acid sequences of a PKSRP and that existwithin a natural population (e.g., a plant species or variety). Suchnatural allelic variations can typically result in 1-5% variance in aPKSRP nucleic acid. Allelic variants can be identified by sequencing thenucleic acid sequence of interest in a number of different plants, whichcan be readily carried out by using hybridization probes to identify thesame PKSRP genetic locus in those plants. Any and all such nucleic acidvariations and resulting amino acid polymorphisms or variations in aPKSRP that are the result of natural allelic variation and that do notalter the functional activity of a PKSRP, are intended to be within thescope of the invention.

Moreover, nucleic acid molecules encoding PKSRPs from the same or otherspecies such as PKSRP analogs, orthologs and paralogs, are intended tobe within the scope of the present invention. As used herein, the term“analogs” refers to two nucleic acids that have the same or similarfunction, but that have evolved separately in unrelated organisms. Asused herein, the term “orthologs” refers to two nucleic acids fromdifferent species, but that have evolved from a common ancestral gene byspeciation. Normally, orthologs encode proteins having the same orsimilar functions. As also used herein, the term “paralogs” refers totwo nucleic acids that are related by duplication within a genome.Paralogs usually have different functions, but these functions may berelated (Tatusov, R. L. et al. 1997 Science 278(5338):631-637). Analogs,orthologs and paralogs of a naturally occurring PKSRP can differ fromthe naturally occurring PKSRP by post-translational modifications, byamino acid sequence differences, or by both. Post-translationalmodifications include in vivo and in vitro chemical derivatization ofpolypeptides, e.g., acetylation, carboxylation, phosphorylation, orglycosylation, and such modifications may occur during polypeptidesynthesis or processing or following treatment with isolated modifyingenzymes. In particular, orthologs of the invention will generallyexhibit at least 80-85%, more preferably 90%, and most preferably 95%,96%, 97%, 98% or even 99% identity or homology with all or part of anaturally occurring PKSRP amino acid sequence and will exhibit afunction similar to a PKSRP. Orthologs of the present invention are alsopreferably capable of participating in the stress response in plants. Inone embodiment, the PKSRP orthologs maintain the ability to participatein the metabolism of compounds necessary for the construction ofcellular membranes in Physcomitrella patens, or in the transport ofmolecules across these membranes.

In addition to naturally-occurring variants of a PKSRP sequence that mayexist in the population, the skilled artisan will further appreciatethat changes can be introduced by mutation into a nucleotide sequence ofSEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:129, therebyleading to changes in the amino acid sequence of the encoded PKSRP,without altering the functional ability of the PKSRP. For example,nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in a sequence of SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:129, or the correspondingamino acid sequences of those disclosed herein. A “non-essential” aminoacid residue is a residue that can be altered from the wild-typesequence of one of the PKSRPs without altering the activity of saidPKSRP, whereas an “essential” amino acid residue is required for PKSRPactivity. Other amino acid residues, however, (e.g., those that are notconserved or only semi-conserved in the domain having PKSRP activity)may not be essential for activity and thus are likely to be amenable toalteration without altering PKSRP activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding PKSRPs that contain changes in amino acid residuesthat are not essential for PKSRP activity. Such PKSRPs differ in aminoacid sequence from a sequence contained in SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,SEQ ID NO:39, SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:135, SEQ IDNO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQID NO:141, SEQ ID NO:142, and SEQ ID NO:143, yet retain at least one ofthe PKSRP activities described herein. In one embodiment, the isolatednucleic acid molecule comprises a nucleotide sequence encoding aprotein, wherein the protein comprises an amino acid sequence at leastabout 50% homologous to an amino acid sequence of SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:3 1, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39,SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:135, SEQ IDNO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQID NO:141, SEQ ID NO:142, and SEQ ID NO:143. Preferably, the proteinencoded by the nucleic acid molecule is at least about 50-60% homologousto one of the sequences of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ IDNO:130, more preferably at least about 60-70% homologous to one of thesequences of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:130, SEQ IDNO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ IDNO:143, even more preferably at least about 70-75%, 75-80%, 80-85%,85-90%, 90-95% homologous to one of the sequences of SEQ ID NO:27, SEQID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:135, SEQ IDNO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ I) NO:140, SEQID NO:141, SEQ ID NO:142, and SEQ ID NO:143, and most preferably atleast about 96%, 97%, 98%, or 99% homologous to one of the sequences ofSEQ ifD NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IfD NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:130, SEQ ID NO:134,SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ IDNO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ I) NO:142, and SEQ ID NO:143.The preferred PKSRP homologs of the present invention are preferablycapable of participating in the a stress tolerance response in a plant,or more particularly, participating in the transcription of a proteininvolved in a stress tolerance response in a Physcomitrella patensplant, or have one or more activities set forth in Table 1.

An isolated nucleic acid molecule encoding a PKSRP homologous to aprotein sequence of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:130, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142,and SEQ ID NO:143 can be created by introducing one or more nucleotidesubstitutions, additions or deletions into a nucleotide sequence of SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:129 such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations can be introduced into one of the sequencesof SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:129 by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain.

Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a PKSRP is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a PKSRP coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor a PKSRP activity described herein to identify mutants that retainPKSRP activity. Following mutagenesis of one of the sequences of SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:]9, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:129, the encoded proteincan be expressed recombinantly and the activity of the protein can bedetermined by analyzing the stress tolerance of a plant expressing theprotein as described in Example 7.

In addition to the nucleic acid molecules encoding the PKSRPs describedabove, another aspect of the invention pertains to isolated nucleic acidmolecules that are antisense thereto. An “antisense” nucleic acidcomprises a nucleotide sequence that is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. Accordingly, an antisense nucleic acid can hydrogen bond to asense nucleic acid. The antisense nucleic acid can be complementary toan entire PKSRP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding a PKSRP.The term “coding region” refers to the region of the nucleotide sequencecomprising codons that are translated into amino acid residues. Inanother embodiment, the antisense nucleic acid molecule is antisense toa “noncoding region” of the coding strand of a nucleotide sequenceencoding a PKSRP. The term “noncoding region” refers to 5′ and 3′sequences that flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of oneof the nucleotide sequences shown in SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26 or SEQ ID NO:129, or a portion thereof. A nucleic acid moleculethat is complementary to one of the nucleotide sequences shown in SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:129 is one which issufficiently complementary to one of the nucleotide sequences shown inSEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:2 1, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:129 such that itcan hybridize to one of the nucleotide sequences shown in SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:129, thereby forming a stableduplex.

Given the coding strand sequences encoding the PKSRPs disclosed herein(e.g., the sequences set forth in SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:2 1, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26 or SEQ ID NO:129), antisense nucleic acids of the invention can bedesigned according to the rules of Watson and Crick base pairing. Theantisense nucleic acid molecule can be complementary to the entirecoding region of PKSRP mRNA, but more preferably is an oligonucleotidewhich is antisense to only a portion of the coding or noncoding regionof PKSRP mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site ofPKSRP mRNA. An antisense oligonucleotide can be, for example, about 5,10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a PKSRP tothereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic (includingplant) promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al., 1987 Nucleic Acids. Res. 15:6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al., 1987 Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987 FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity, which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes described in Haselhoff andGerlach, 1988 Nature 334:585-591) can be used to catalytically cleavePKSRP mRNA transcripts to thereby inhibit translation of PKSRP mRNA. Aribozyme having specificity for a PKSRP-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a PKSRP cDNA, asdisclosed herein (i.e., SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 SEQ ID orNO:129) or on the basis of a heterologous sequence to be isolatedaccording to methods taught in this invention. For example, a derivativeof a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a PKSRP-encoding mRNA. See, e.g., Cech et al. U.S. Pat.No. 4,987,071 and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,PKSRP mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel,D. and Szostak, J. W., 1993 Science 261:1411-1418.

Alternatively, PKSRP gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of a PKSRPnucleotide sequence (e.g., a PKSRP promoter and/or enhancer) to formtriple helical structures that prevent transcription of a PKSRP gene intarget cells. See generally, Helene, C., 1991 Anticancer Drug Des.6(6):569-84; Helene, C. et al., 1992 Ann. N.Y. Acad. Sci. 660:27-36; andMaher, L. J., 1992 Bioassays 14(12):807-15.

In addition to the PKSRP nucleic acids and proteins described above, thepresent invention encompasses these nucleic acids and proteins attachedto a moiety. These moieties include, but are not limited to, detectionmoieties, hybridization moieties, purification moieties, deliverymoieties, reaction moieties, binding moieties, and the like. A typicalgroup of nucleic acids having moieties attached are probes and primers.The probes and primers typically comprise a substantially isolatedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 40, 50 or 75consecutive nucleotides of a sense strand of one of the sequences setforth in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:129, ananti-sense sequence of one of the sequences set forth in SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:129, or naturally occurringmutants thereof. Primers based on a nucleotide sequence of SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:129 can be used in PCR reactionsto clone PKSRP homologs. Probes based on the PKSRP nucleotide sequencescan be used to detect transcripts or genomic sequences encoding the sameor homologous proteins. In preferred embodiments, the probe furthercomprises a label group attached thereto, e.g. the label group can be aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as a part of a genomic marker test kit foridentifying cells which express a PKSRP, such as by measuring a level ofa PKSRP-encoding nucleic acid, in a sample of cells, e.g., detectingPKSRP mRNA levels or determining whether a genomic PKSRP gene has beenmutated or deleted.

In particular, a useful method to ascertain the level of transcriptionof the gene (an indicator of the amount of mRNA available fortranslation to the gene product) is to perform a Northern blot (forreference see, for example, Ausubel et al., 1988 Current Protocols inMolecular Biology, Wiley: New York). This information at least partiallydemonstrates the degree of transcription of the transformed gene. Totalcellular RNA can be prepared from cells, tissues or organs by severalmethods, all well-known in the art, such as that described in Bormann,E. R. et al., 1992 Mol. Microbiol. 6:317-326. To assess the presence orrelative quantity of protein translated from this mRNA, standardtechniques, such as a Western blot, may be employed. These techniquesare well known to one of ordinary skill in the art. (See, for example,Ausubel et al., 1988 Current Protocols in Molecular Biology, Wiley: NewYork).

The invention further provides an isolated recombinant expression vectorcomprising a PKSRP nucleic acid as described above, wherein expressionof the vector in a host cell results in increased tolerance toenvironmental stress as compared to a wild type variety of the hostcell. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an iiivitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990) or see:Gruber and Crosby, in: Methods in Plant Molecular Biology andBiotechnology, eds. Glick and Thompson, Chapter 7, 89-108, CRC Press:Boca Raton, Fla., including the references therein. Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells and those that direct expression ofthe nucleotide sequence only in certain host cells or under certainconditions. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein (e.g., PKSRPs, mutant forms of PKSRPs, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of PKSRPs in prokaryotic or eukaryotic cells. For example,PKSRP genes can be expressed in bacterial cells such as C. glutamicum,insect cells (using baculovirus expression vectors), yeast and otherfungal cells (see Romanos, M. A. et al., 1992 Foreign gene expression inyeast: a review, Yeast 8:423-488; van den Hondel, C. A. M. J. J. et al.,1991 Heterologous gene expression in filamentous fungi, in: More GeneManipulations in Fungi, J. W. Bennet & L. L. Lasure, eds., p. 396-428:Academic Press: San Diego; and van den Hondel, C. A. M. J. J. & Punt, P.J., 1991 Gene transfer systems and vector development for filamentousfungi, in: Applied Molecular Genetics of Fungi, Peberdy, J. F. et al.,eds., p. 1-28, Cambridge University Press: Cambridge), algae (Falciatoreet al., 1999 Marine Biotechnology 1(3):239-251), ciliates of the types:Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena,Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyof the genus Stylonychia lemnae with vectors following a transformationmethod as described in WO 98/01572 and multicellular plant cells (seeSchmidt, R. and Willmitzer, L., 1988 High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants, Plant Cell Rep. 583-586); Plant Molecular Biologyand Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, S.71-119(1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in:Transgenic Plants, Vol. 1, Engineering and Utilization, eds. Kung und R.Wu, 128-43, Academic Press: 1993; Potrykus, 1991 Annu. Rev. PlantPhysiol. Plant Molec. Biol. 42:205-225 and references cited therein) ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press:San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, usually to the aminoterminus of the recombinant protein but also to the C-terminus or fusedwithin suitable regions in the proteins. Such fusion vectors typicallyserve three purposes: 1) to increase expression of a recombinantprotein; 2) to increase the solubility of a recombinant protein; and 3)to aid in the purification of a recombinant protein by acting as aligand in affinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S., 1988 Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the PKSRP is cloned into a pGEXexpression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-X protein. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant PKSRPunfused to GST can be recovered by cleavage of the fusion protein withthrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988 Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by aco-expressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS 174(DE3) from a resident Xprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression is to expressthe protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gollesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the sequenceof the nucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin the bacterium chosen for expression, such as C. glutamicum (Wada etal., 1992 Nucleic Acids Res. 20:2111-2118). Such alteration of nucleicacid sequences of the invention can be carried out by standard DNAsynthesis techniques.

In another embodiment, the PKSRP expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., 1987 Embo J. 6:229-234), pMFa (Kujanand Herskowitz, 1982 Cell 30:933-943), pJRY88 (Schultz et al., 1987 Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).Vectors and methods for the construction of vectors appropriate for usein other fungi, such as the filamentous fungi, include those detailedin: van den Hondel, C. A. M. J. J. & Punt, P. J. (1991) “Gene transfersystems and vector development for filamentous fungi, in: AppliedMolecular Genetics of Fungi, J. F. Peberdy, et al., eds., p. 1-28,Cambridge University Press: Cambridge.

Alternatively, the PKSRPs of the invention can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., 1983 Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers, 1989 Virology170:31-39).

In yet another embodiment, a PKSRP nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B., 1987Nature 329:840) and pMT2PC (Kaufman et al., 1987 EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2^(nd) ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.,1987 Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton, 1988 Adv. Immunol. 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore, 1989 EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., 1983 Cell 33:729-740; Queen andBaltimore, 1983 Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989 PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al., 1985 Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for example,the murine hox promoters (Kessel and Gruss, 1990 Science 249:374-379)and the fetoprotein promoter (Campes and Tilghman, 1989 Genes Dev.3:537-546).

In another embodiment, the PKSRPs of the invention may be expressed inunicellular plant cells (such as algae) (see Falciatore et al., 1999Marine Biotechnology 1(3):239-251 and references therein) and plantcells from higher plants (e.g., the spermatophytes, such as cropplants). Examples of plant expression vectors include those detailed in:Becker, D., Kemper, E., Schell, J. and Masterson, R., 1992 New plantbinary vectors with selectable markers located proximal to the leftborder, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W., 1984 BinaryAgrobacterium vectors for plant transformation, Nucl. Acid. Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, eds.: Kung and R. Wu,Academic Press, 1993, S. 15-38.

A plant expression cassette preferably contains regulatory sequencescapable of driving gene expression in plant cells and operably linked sothat each sequence can fulfill its function, for example, termination oftranscription by polyadenylation signals. Preferred polyadenylationsignals are those originating from Agrobacterium tumefaciens t-DNA suchas the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5(Gielen et al., 1984 EMBO J. 3:835) or functional equivalents thereofbut also all other terminators functionally active in plants aresuitable.

As plant gene expression is very often not limited on transcriptionallevels, a plant expression cassette preferably contains other operablylinked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al.,1987 Nucl. Acids Research 15:8693-8711).

Plant gene expression has to be operably linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Preferred are promoters driving constitutive expression (Benfeyet al., 1989 EMBO J. 8:2195-2202) like those derived from plant viruseslike the 35S CAMV (Franck et al., 1980 Cell 21:285-294), the 19S CaMV(see also U.S. Pat. No. 5352605 and PCT Application No. WO 8402913) orplant promoters like those from Rubisco small subunit described in U.S.Pat. No. 4,962,028.

Other preferred sequences for use in plant gene expression cassettes aretargeting-sequences necessary to direct the gene product in itsappropriate cell compartment (for review see Kermode, 1996 Crit. Rev.Plant Sci. 15(4):285-423 and references cited therein) such as thevacuole, the nucleus, all types of plastids like amyloplasts,chloroplasts, chromoplasts, the extracellular space, mitochondria, theendoplasmic reticulum, oil bodies, peroxisomes and other compartments ofplant cells.

Plant gene expression can also be facilitated via an inducible promoter(for review see Gatz, 1997 Annu. Rev. Plant Physiol. Plant Mol. Biol.48:89-108). Chemically inducible promoters are especially suitable ifgene expression is wanted to occur in a time specific manner. Examplesof such promoters are a salicylic acid inducible promoter (PCTApplication No. WO 95/19443), a tetracycline inducible promoter (Gatz etal., 1992 Plant J. 2:397-404) and an ethanol inducible promoter (PCTApplication No. WO 93/21334).

Also, suitable promoters responding to biotic or abiotic stressconditions are those such as the pathogen inducible PRP1-gene promoter(Ward et al., 1993 Plant. Mol. Biol. 22:361-366), the heat induciblehsp80-promoter from tomato (U.S. Pat. No. 5187267), cold induciblealpha-amylase promoter from potato (PCT Application No. WO 96/12814) orthe wound-inducible pinII-promoter (European Patent No. 375091). Forother examples of drought, cold, and salt-inducible promoters, such asthe RD29A promoter, see Yamaguchi-Shinozalei et al. (1993 Mol. Gen.Genet. 236:331-340).

Especially preferred are those promoters that confer gene expression inspecific tissues and organs, such as guard cells and the root haircells. Suitable promoters include the napin-gene promoter from rapeseed(U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba (Baeumleinet al., 1991 Mol Gen Genet. 225(3):459-67), the oleosin-promoter fromArabidopsis (PCT Application No. WO 98/45461), the phaseolin-promoterfrom Phaseolus vulgaris (U.S. Patent No. 5,504,200), the Bce4-promoterfrom Brassica (PCT Application No. WO 91/13980) or the legumin B4promoter (LeB4; Baeumlein et al., 1992 Plant Journal, 2(2):233-9) aswell as promoters conferring seed specific expression in monocot plantslike maize, barley, wheat, rye, rice, etc. Suitable promoters to noteare the 1pt2 or 1pt1-gene promoter from barley (PCT Application No. WO95/15389 and PCT Application No. WO 95/23230) or those described in PCTApplication No. WO 99/16890 (promoters from the barley hordein-gene,rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadingene, wheat glutelin gene, maize zein gene, oat glutelin gene, Sorghumkasirin-gene and rye secalin gene).

Also especially suited are promoters that confer plastid-specific geneexpression since plastids are the compartment where lipid biosynthesisoccurs. Suitable promoters are the viral RNA-polymerase promoterdescribed in PCT Application No. WO 95/16783 and PCT Application No. WO97/06250 and the clpP-promoter from Arabidopsis described in PCTApplication No. WO 99/46394.

The invention further provides a recombinant expression vectorcomprising a PKSRP DNA molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner that allowsfor expression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to a PKSRP mRNA. Regulatory sequences operativelylinked to a nucleic acid molecule cloned in the antisense orientationcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types. For instance, viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus wherein antisensenucleic acids are produced under the control of a high efficiencyregulatory region. The activity of the regulatory region can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub, H. et al., Antisense RNA as a molecular tool for geneticanalysis, Reviews—Trends in Genetics, Vol. 1(1) 1986 and Mol et al.,1990 FEBS Letters 268:427-430.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but they also apply to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aPKSRP can be expressed in bacterial cells such as C. glutamicum, insectcells, fungal cells or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells), algae, ciliates, plant cells, fungi or othermicroorganisms like C. gluatamicum. Other suitable host cells are knownto those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation”, “transfection”, “conjugation” and“transduction” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer and electroporation. Suitable methods fortransforming or transfecting host cells including plant cells can befound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual.2^(nd,) ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) and other laboratorymanuals such as Methods in Molecular Biology, 1995, Vol. 44,Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa,N.J. As biotic and abiotic stress tolerance is a general trait wished tobe inherited into a wide variety of plants like maize, wheat, rye, oat,triticale, rice, barley, soybean, peanut, cotton, rapeseed and canola,manihot, pepper, sunflower and tagetes, solanaceous plants like potato,tobacco, eggplant, and tomato, Vicia species, pea, alfalfa, bushy plants(coffee, cacao, tea), Salix species, trees (oil palm, coconut),perennial grasses and forage crops, these crop plants are also preferredtarget plants for a genetic engineering as one further embodiment of thepresent invention.

In particular, the invention provides a method of producing a transgenicplant with a PKSRP coding nucleic acid, wherein expression of thenucleic acid(s) in the plant results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcomprising: (a) transforming a plant cell with an expression vectorcomprising a PKSRP nucleic acid, and (b) generating from the plant cella transgenic plant with a increased tolerance to environmental stress ascompared to a wild type variety of the plant. The invention alsoprovides a method of increasing expression of a gene of interest withina host cell as compared to a wild type variety of the host cell, whereinthe gene of interest is transcribed in response to a PKSRP, comprising:(a) transforming the host cell with an expression vector comprising aPKSRP coding nucleic acid, and (b) expressing the PKSRP within the hostcell, thereby increasing the expression of the gene transcribed inresponse to the PKSRP, as compared to a wild type variety of the hostcell.

For such plant transformation, binary vectors such as pBinAR can be used(Höfgen and Willmitzer, 1990 Plant Science 66:221-230). Construction ofthe binary vectors can be performed by ligation of the cDNA in sense orantisense orientation into the T-DNA. 5-prime to the cDNA a plantpromoter activates transcription of the cDNA. A polyadenylation sequenceis located 3-prime to the cDNA. Tissue-specific expression can beachieved by using a tissue specific promoter. For example, seed-specificexpression can be achieved by cloning the napin or LeB4 or USP promoter5-prime to the cDNA. Also, any other seed specific promoter element canbe used. For constitutive expression within the whole plant, the CaMV35S promoter can be used. The expressed protein can be targeted to acellular compartment using a signal peptide, for example for plastids,mitochondria or endoplasmic reticulum (Kermode, 1996 Crit. Rev. PlantSci. 4(15):285-423). The signal peptide is cloned 5-prime in frame tothe cDNA to archive subcellular localization of the fusion protein.Additionally, promoters that are responsive to abiotic stresses can beused with, such as the Arabidopsis promoter RD29A, the nucleic acidsequences disclosed herein. One skilled in the art will recognize thatthe promoter used should be operatively linked to the nucleic acid suchthat the promoter causes transcription of the nucleic acid which resultsin the synthesis of a mRNA which encodes a polypeptide. Alternatively,the RNA can be an antisense RNA for use in affecting subsequentexpression of the same or another gene or genes.

Alternate methods of transfection include the direct transfer of DNAinto developing flowers via electroporation or Agrobacterium mediatedgene transfer. Agrobacterium mediated plant transformation can beperformed using for example the GV3101(pMP90) (Koncz and Schell, 1986Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacteriumtumefaciens strain. Transformation can be performed by standardtransformation and regeneration techniques (Deblaere et al., 1994 Nucl.Acids. Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A,Plant Molecular Biology Manual, 2^(nd) Ed.—Dordrecht: Kluwer AcademicPubl., 1995.—in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN0-7923-2731-4; Glick, Bernard R.; Thompson, John E., Methods in PlantMolecular Biology and Biotechnology, Boca Raton : CRC Press, 1993.-360S., ISBN 0-8493-5164-2). For example, rapeseed can be transformed viacotyledon or hypocotyl transformation (Moloney et al., 1989 Plant cellReport 8:238-242; De Block et al., 1989 Plant Physiol. 91:694-701). Useof antibiotica for Agrobacterium and plant selection depends on thebinary vector and the Agrobacterium strain used for transformation.Rapeseed selection is normally performed using kanamycin as selectableplant marker. Agrobacterium mediated gene transfer to flax can beperformed using, for example, a technique described by Mlynarova et al.,1994 Plant Cell Report 13:282-285. Additionally, transformation ofsoybean can be performed using for example a technique described inEuropean Patent No. 0424 047, U.S. Pat. No. 5,322,783, European PatentNo. 0397 687, U.S. Pat. No. 5,376,543 or U.S. Pat. No. 5,169,770.Transformation of maize can be achieved by particle bombardment,polyethylene glycol mediated DNA uptake or via the silicon carbide fibertechnique. (See, for example, Freeling and Walbot “The maize handbook”Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific exampleof maize transformation is found in U.S. Pat. No. 5,990,387 and aspecific example of wheat transformation can be found in PCT ApplicationNo. WO 93/07256.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate or in plants thatconfer resistance towards a herbicide such as glyphosate or glufosinate.Nucleic acid molecules encoding a selectable marker can be introducedinto a host cell on the same vector as that encoding a PKSRP or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid molecule can be identified by, for example, drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

To create a homologous recombinant microorganism, a vector is preparedwhich contains at least a portion of a PKSRP gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the PKSRP gene. Preferably, the PKSRP gene is aPhyscomitrella patens PKSRP gene, but it can be a homolog from a relatedplant or even from a mammalian, yeast, or insect source. In a preferredembodiment, the vector is designed such that, upon homologousrecombination, the endogenous PKSRP gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as aknock-out vector). Alternatively, the vector can be designed such that,upon homologous recombination, the endogenous PKSRP gene is mutated orotherwise altered but still encodes a functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous PKSRP). To create a point mutation viahomologous recombination, DNA-RNA hybrids can be used in a techniqueknown as chimeraplasty (Cole-Strauss et al., 1999 Nucleic Acids Research27(5):1323-1330 and Kmiec, 1999 Gene therapy American Scientist.87(3):240-247). Homologous recombination procedures in Physcomitrellapatens are also well known in the art and are contemplated for useherein.

Whereas in the homologous recombination vector, the altered portion ofthe PKSRP gene is flanked at its 5′ and 3′ ends by an additional nucleicacid molecule of the PKSRP gene to allow for homologous recombination tooccur between the exogenous PKSRP gene carried by the vector and anendogenous PKSRP gene, in a microorganism or plant. The additionalflanking PKSRP nucleic acid molecule is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several hundreds of base pairs up to kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see e.g., Thomas, K. R.,and Capecchi, M. R., 1987 Cell 51:503 for a description of homologousrecombination vectors or Strepp et al., 1998 PNAS, 95 (8):4368-4373 forcDNA based recombination in Physcomitrella patens). The vector isintroduced into a microorganism or plant cell (e.g., via polyethyleneglycol mediated DNA), and cells in which the introduced PKSRP gene hashomologously recombined with the endogenous PKSRP gene are selectedusing art-known techniques.

In another embodiment, recombinant microorganisms can be produced thatcontain selected systems which allow for regulated expression of theintroduced gene. For example, inclusion of a PKSRP gene on a vectorplacing it under control of the lac operon permits expression of thePKSRP gene only in the presence of IPTG. Such regulatory systems arewell known in the art.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a PKSRP.Accordingly, the invention further provides methods for producing PKSRPsusing the host cells of the invention. In one embodiment, the methodcomprises culturing the host cell of invention (into which a recombinantexpression vector encoding a PKSRP has been introduced, or into whichgenome has been introduced a gene encoding a wild-type or altered PKSRP) in a suitable medium until PKSRP is produced. In another embodiment,the method further comprises isolating PKSRPs from the medium or thehost cell.

Another aspect of the invention pertains to isolated PKSRPs, andbiologically active portions thereof. An “isolated” or “purified”protein or biologically active portion thereof is free of some of thecellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof PKSRP in which the protein is separated from some of the cellularcomponents of the cells in which it is naturally or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of a PKSRP having less thanabout 30% (by dry weight) of non-PKSRP material (also referred to hereinas a “contaminating protein”), more preferably less than about 20% ofnon-PKSRP material, still more preferably less than about 10% ofnon-PKSRP material, and most preferably less than about 5% non-PKSRPmaterial.

When the PKSRP or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation. The language “substantially free ofchemical precursors or other chemicals” includes preparations of PKSRPin which the protein is separated from chemical precursors or otherchemicals that are involved in the synthesis of the protein. In oneembodiment, the language “substantially free of chemical precursors orother chemicals” includes preparations of a PKSRP having less than about30% (by dry weight) of chemical precursors or non-PKSRP chemicals, morepreferably less than about 20% chemical precursors or non-PKSRPchemicals, still more preferably less than about 10% chemical precursorsor non-PKSRP chemicals, and most preferably less than about 5% chemicalprecursors or non-PKSRP chemicals. In preferred embodiments, isolatedproteins, or biologically active portions thereof, lack contaminatingproteins from the same organism from which the PKSRP is derived.Typically, such proteins are produced by recombinant expression of, forexample, a Phycomitrella patens PKSRP in plants other thanPhyscomitrella patens or microorganisms such as C. glutamicum, ciliates,algae or fungi.

The nucleic acid molecules, proteins, protein homologs, fusion proteins,primers, vectors, and host cells described herein can be used in one ormore of the following methods: identification of Physcomitrella patensand related organisms; mapping of genomes of organisms related toPhyscomitrella patens; identification and localization of Physcomitrellapatens sequences of interest; evolutionary studies; determination ofPKSRP regions required for function; modulation of a PKSRP activity;modulation of the metabolism of one or more cell functions; modulationof the transmembrane transport of one or more compounds; and modulationof stress resistance.

The moss Physcomitrella patens represents one member of the mosses. Itis related to other mosses such as Ceratodon purpureus which is capableof growth in the absence of light. Mosses like Ceratodon andPhyscomitrella share a high degree of homology on the DNA sequence andpolypeptide level allowing the use of heterologous screening of DNAmolecules with probes evolving from other mosses or organisms, thusenabling the derivation of a consensus sequence suitable forheterologous screening or functional annotation and prediction of genefunctions in third species. The ability to identify such functions cantherefore have significant relevance, e.g., prediction of substratespecificity of enzymes. Further, these nucleic acid molecules may serveas reference points for the mapping of moss genomes, or of genomes ofrelated organisms.

The PKSRP nucleic acid molecules of the invention have a variety ofuses. Most importantly, the nucleic acid and amino acid sequences of thepresent invention can be used to transform plants, thereby inducingtolerance to stresses such as drought, high salinity and cold. Thepresent invention therefore provides a transgenic plant transformed by aPKSRP nucleic acid (coding or antisense), wherein expression of thenucleic acid sequence in the plant results in increased tolerance toenvironmental stress as compared to a wild type variety of the plant.The transgenic plant can be a monocot or a dicot. The invention furtherprovides that the transgenic plant can be selected from maize, wheat,rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed,canola, manihot, pepper, sunflower, tagetes, solanaceous plants, potato,tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao,tea, Salix species, oil palm, coconut, perennial grass and forage crops,for example.

In particular, the present invention describes using the expression ofPK-6, PK-7, PK-8, PK-9, CK-1, CK-2, CK-3, CK2-1, MPK-2, MPK-3, MPK4,MPK-5, CPK-1 and CPK-2 of Physcomitrella patens to engineerdrought-tolerant, salt-tolerant and/or cold-tolerant plants. Thisstrategy has herein been demonstrated for Arabidopsis thaliana,Rapeseed/Canola, soybeans, corn and wheat but its application is notrestricted to these plants. Accordingly, the invention provides atransgenic plant containing a PKSRP selected from PK-6 (SEQ ID NO:27),PK-7 (SEQ ID NO:28), PK-8 (SEQ ID NO:29), PK-9 (SEQ ID NO:30), CK-1 (SEQID NO:31), CK-2 (SEQ ID NO:32), CK-3 (SEQ ID NO:33), MPK-2 (SEQ IDNO:34), MPK-3 (SEQ ID NO:35), MPK-4 (SEQ ID NO:36), MPK-5 (SEQ IDNO:37), CPK-1 (SEQ ID NO:38), CPK-2 (SEQ ID NO:39), and CK2-1 (SEQ IDNO:130) and those comprising SEQ ID NO:134, SEQ ID NO:135, SEQ IDNO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQID NO:141, SEQ ID NO:142, and SEQ ID NO:143, wherein the environmentalstress is drought, increased salt or decreased or increased temperature.In preferred embodiments, the environmental stress is drought ordecreased temperature.

The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a PKSRP inthe plant. The invention provides that this method can be performed suchthat the stress tolerance is either increased or decreased. Inparticular, the present invention provides methods of producing atransgenic plant having an increased tolerance to environmental stressas compared to a wild type variety of the plant comprising increasingexpression of a PKSRP in a plant.

The methods of increasing expression of PKSRPs can be used wherein theplant is either transgenic or not transgenic. In cases when the plant istransgenic, the plant can be transformed with a vector containing any ofthe above described PKSRP coding nucleic acids, or the plant can betransformed with a promoter that directs expression of native PKSRP inthe plant, for example. The invention provides that such a promoter canbe tissue specific. Furthermore, such a promoter can be developmentallyregulated. Alternatively, non-transgenic plants can have native PKSRPexpression modified by inducing a native promoter.

The expression of PK-6 (SEQ ID NO:14), PK-7 (SEQ ID NO:15), PK-8 (SEQ IDNO:16), PK-9 (SEQ ID NO:17), CK-1 (SEQ ID NO:18), CK-2 (SEQ ID NO:19),CK-3 (SEQ ID NO:20), MPK-2 (SEQ ID NO:21), MPK-3 (SEQ ID NO:22), MPK-4(SEQ ID NO:23), MPK-5 (SEQ ID NO:24), CPK-1 (SEQ ID NO:25), CPK-2 (SEQID NO:26) and CK2-1 (SEQ ID NO:129) in target plants can be accomplishedby, but is not limited to, one of the following examples: (a)constitutive promoter, (b) stress-inducible promoter, (c)chemical-induced promoter, and (d) engineered promoter over-expressionwith for example zinc-finger derived transcription factors (Greisman andPabo, 1997 Science 275:657). The later case involves identification ofthe PK-6 (SEQ ID NO:27), PK-7 (SEQ ID NO:28), PK-8 (SEQ ID NO:29), PK-9(SEQ ID NO:30), CK-1 (SEQ ID NO:31), CK-2 (SEQ ID NO:32), CK-3 (SEQ IDNO:33), MPK-2 (SEQ ID NO:34), MPK-3 (SEQ ID NO:35), MPK-4 (SEQ IDNO:36), MPK-5 (SEQ ID NO:37), CPK-1 (SEQ ID NO:38), CPK-2 (SEQ ID NO:39)or CK2-1 (SEQ ID NO:130) and those comprising SEQ ID NO:134, SEQ IDNO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143 homologs inthe target plant as well as from its promoter. Zinc-finger-containingrecombinant transcription factors are engineered to specificallyinteract with the PK-6 (SEQ ID NO:27), PK-7 (SEQ ID NO:28), PK-8 (SEQ IDNO:29), PK-9 (SEQ ID NO:30), CK-1 (SEQ ID NO:31), CK-2 (SEQ ID NO:32),CK-3 (SEQ ID NO:33), MPK-2 (SEQ ID NO:34), MPK-3 (SEQ ID NO:35), MPK-4(SEQ ID NO:36), MPK-5 (SEQ ID NO:37), CPK-1 (SEQ If NO:38), CPK-2 (SEQID NO:39) or CK2-1 (SEQ ID NO:130) and those comprising SEQ ID NO:134,SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ IDNO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143homolog and transcription of the corresponding gene is activated.

In addition to introducing the PKSRP nucleic acid sequences intotransgenic plants, these sequences can also be used to identify anorganism as being Phycomitrella patens or a close relative thereof Also,they may be used to identify the presence of Physcomitrella patens or arelative thereof in a mixed population of microorganisms. The inventionprovides the nucleic acid sequences of a number of Physcomitrella patensgenes; by probing the extracted genomic DNA of a culture of a unique ormixed population of microorganisms under stringent conditions with aprobe spanning a region of a Physcomitrella patens gene which is uniqueto this organism, one can ascertain whether this organism is present.

Further, the nucleic acid and protein molecules of the invention mayserve as markers for specific regions of the genome. This has utilitynot only in the mapping of the genome, but also in functional studies ofPhyscomitrella patens proteins. For example, to identify the region ofthe genome to which a particular Physcomitrella patens DNA-bindingprotein binds, the Physcomitrella patens genome could be digested, andthe fragments incubated with the DNA-binding protein. Those fragmentsthat bind the protein may be additionally probed with the nucleic acidmolecules of the invention, preferably with readily detectable labels.Binding of such a nucleic acid molecule to the genome fragment enablesthe localization of the fragment to the genome map of Physcomitrellapatens, and, when performed multiple times with different enzymes,facilitates a rapid determination of the nucleic acid sequence to whichthe protein binds. Further, the nucleic acid molecules of the inventionmay be sufficiently homologous to the sequences of related species suchthat these nucleic acid molecules may serve as markers for theconstruction of a genomic map in related mosses.

The PKSRP nucleic acid molecules of the invention are also useful forevolutionary and protein structural studies. The metabolic and transportprocesses in which the molecules of the invention participate areutilized by a wide variety of prokaryotic and eukaryotic cells; bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar enzymes from other organisms, theevolutionary relatedness of the organisms can be assessed. Similarly,such a comparison permits an assessment of which regions of the sequenceare conserved and which are not, which may aid in determining thoseregions of the protein that are essential for the functioning of theenzyme. This type of determination is of value for protein engineeringstudies and may give an indication of what the protein can tolerate interms of mutagenesis without losing function.

Manipulation of the PKSRP nucleic acid molecules of the invention mayresult in the production of PKSRPs having functional differences fromthe wild-type PKSRPs. These proteins may be improved in efficiency oractivity, may be present in greater numbers in the cell than is usual,or may be decreased in efficiency or activity.

There are a number of mechanisms by which the alteration of a PKSRP ofthe invention may directly affect stress response and/or stresstolerance. In the case of plants expressing PKSRPs, increased transportcan lead to improved salt and/or solute partitioning within the planttissue and organs. By either increasing the number or the activity oftransporter molecules which export ionic molecules from the cell, it maybe possible to affect the salt tolerance of the cell.

The effect of the genetic modification in plants, C. glutamicum, fungi,algae, or ciliates on stress tolerance can be assessed by growing themodified microorganism or plant under less than suitable conditions andthen analyzing the growth characteristics and/or metabolism of theplant. Such analysis techniques are well known to one skilled in theart, and include dry weight, wet weight, protein synthesis, carbohydratesynthesis, lipid synthesis, evapotranspiration rates, general plantand/or crop yield, flowering, reproduction, seed setting, root growth,respiration rates, photosynthesis rates, etc. (Applications of HPLC inBiochemistry in: Laboratory Techniques in Biochemistry and MolecularBiology, vol. 17; Rehm et al., 1993 Biotechnology, vol. 3, Chapter III:Product recovery and purification, page 469-714, VCH: Weinheim; Belter,P. A. et al., 1988 Bioseparations: downstream processing forbiotechnology, John Wiley and Sons; Kennedy, J. F. and Cabral, J. M. S.,1992 Recovery processes for biological materials, John Wiley and Sons;Shaeiwitz, J. A. and Henry, J. D., 1988 Biochemical separations, in:Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation andpurification techniques in biotechnology, Noyes Publications).

For example, yeast expression vectors comprising the nucleic acidsdisclosed herein, or fragments thereof, can be constructed andtransformed into Saccharomyces cerevisiae using standard protocols. Theresulting transgenic cells can then be assayed for fail or alteration oftheir tolerance to drought, salt, and temperature stress. Similarly,plant expression vectors comprising the nucleic acids disclosed herein,or fragments thereof, can be constructed and transformed into anappropriate plant cell such as Arabidopsis, soy, rape, maize, wheat,Medicago truncatula, etc., using standard protocols. The resultingtransgenic cells and/or plants derived there from can then be assayedfor fail or alteration of their tolerance to drought, salt, andtemperature stress.

The engineering of one or more PKSRP genes of the invention may alsoresult in PKSRPs having altered activities which indirectly impact thestress response and/or stress tolerance of algae, plants, ciliates orfungi or other microorganisms like C. glutamicum. For example, thenormal biochemical processes of metabolism result in the production of avariety of products (e.g., hydrogen peroxide and other reactive oxygenspecies) which may actively interfere with these same metabolicprocesses (for example, peroxynitrite is known to nitrate tyrosine sidechains, thereby inactivating some enzymes having tyrosine in the activesite (Groves, J. T., 1999 Curr. Opin. Chem. Biol. 3(2):226-235). Whilethese products are typically excreted, cells can be genetically alteredto transport more products than is typical for a wild-type cell. Byoptimizing the activity of one or more PKSRPs of the invention which areinvolved in the export of specific molecules, such as salt molecules, itmay be possible to improve the stress tolerance of the cell.

Additionally, the sequences disclosed herein, or fragments thereof, canbe used to generate knockout mutations in the genomes of variousorganisms, such as bacteria, mammalian cells, yeast cells, and plantcells (Girke, T., 1998 The Plant Journal 15:39-48). The resultantknockout cells can then be evaluated for their ability or capacity totolerate various stress conditions, their response to various stressconditions, and the effect on the phenotype and/or genotype of themutation. For other methods of gene inactivation see U.S. Pat. No.6004804 “Non-Chimeric Mutational Vectors” and Puttaraju et al., 1999Spliceosome-mediated RNA trans-splicing as a tool for gene therapyNature Biotechnology 17:246-252.

The aforementioned mutagenesis strategies for PKSRPs resulting inincreased stress resistance are not meant to be limiting; variations onthese strategies will be readily apparent to one skilled in the art.Using such strategies, and incorporating the mechanisms disclosedherein, the nucleic acid and protein molecules of the invention may beutilized to generate algae, ciliates, plants, fungi or othermicroorganisms like C. glutamicum expressing mutated PKSRP nucleic acidand protein molecules such that the stress tolerance is improved.

The present invention also provides antibodies that specifically bind toa PKSRP, or a portion thereof, as encoded by a nucleic acid describedherein. Antibodies can be made by many well-known methods (See, e.g.Harlow and Lane, “Antibodies; A Laboratory Manual” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigencan be injected into an animal in an amount and in intervals sufficientto elicit an immune response. Antibodies can either be purifieddirectly, or spleen cells can be obtained from the animal. The cells canthen fused with an immortal cell line and screened for antibodysecretion. The antibodies can be used to screen nucleic acid clonelibraries for cells secreting the antigen. Those positive clones canthen be sequenced. (See, for example, Kelly et al., 1992 Bio/Technology10:163-167; Bebbington et al., 1992 Bio/Technology 10:169-175).

The phrases “selectively binds” and “specifically binds” with thepolypeptide refer to a binding reaction that is determinative of thepresence of the protein in a heterogeneous population of proteins andother biologics. Thus, under designated immunoassay conditions, thespecified antibodies bound to a particular protein do not bind in asignificant amount to other proteins present in the sample. Selectivebinding of an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. A varietyof immunoassay formats may be used to select antibodies that selectivelybind with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies selectivelyimmunoreactive with a protein. See Harlow and Lane “Antibodies, ALaboratory Manual” Cold Spring Harbor Publications, New York, (1988),for a description of immunoassay formats and conditions that could beused to determine selective binding.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious hosts. A description of techniques for preparing such monoclonalantibodies may be found in Stites et al., editors, “Basic and ClinicalImmunology,” (Lange Medical Publications, Los Altos, Calif., FourthEdition) and references cited therein, and in Harlow and Lane(“Antibodies, A Laboratory Manual” Cold Spring Harbor Publications, NewYork, 1988).

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

It should also be understood that the foregoing relates to preferredembodiments of the present invention and that numerous changes may bemade therein without departing from the scope of the invention. Theinvention is further illustrated by the following examples, which arenot to be construed in any way as imposing limitations upon the scopethereof On the contrary, it is to be clearly understood that resort maybe had to various other embodiments, modifications, and equivalentsthereof, which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the present invention and/or the scope of the appended claims.

EXAMPLES Example 1

Growth of Physcomitrella patens Cultures

For this study, plants of the species Physcomitrella patens (Hedw.)B.S.G. from the collection of the genetic studies section of theUniversity of Hamburg were used. They originate from the strain 16/14collected by H. L. K. Whitehouse in Gransden Wood, Huntingdonshire(England), which was subcultured from a spore by Engel (1968, Am. J.Bot. 55, 438-446). Proliferation of the plants was carried out by meansof spores and by means of regeneration of the gametophytes. Theprotonema developed from the haploid spore as a chloroplast-richchloronema and chloroplast-low caulonema, on which buds formed afterapproximately 12 days. These grew to give gametophores bearingantheridia and archegonia. After fertilization, the diploid sporophytewith a short seta and the spore capsule resulted, in which themeiospores matured.

Culturing was carried out in a climatic chamber at an air temperature of25° C. and light intensity of 55 micromols⁻¹m²(white light; Philips TL65W/25 fluorescent tube) and a light/dark change of 16/8 hours. The mosswas either modified in liquid culture using Knop medium according toReski and Abel (1985, Planta 165:354-358) or cultured on Knop solidmedium using 1% oxoid agar (Unipath, Basingstoke, England). Theprotonemas used for RNA and DNA isolation were cultured in aeratedliquid cultures. The protonemas were comminuted every 9 days andtransferred to fresh culture medium.

Example 2

Total DNA Isolation from Plants

The details for the isolation of total DNA relate to the working up ofone gram fresh weight of plant material. The materials used include thefollowing buffers: CTAB buffer: 2% (w/v)N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCl pH 8.0;1.4 M NaCl; 20 mM EDTA; N-Laurylsarcosine buffer: 10% (w/v)N-laurylsarcosine; 100 mM Tris HCl pH 8.0; 20 mM EDTA.

The plant material was triturated under liquid nitrogen in a mortar togive a fine powder and transferred to 2 ml Eppendorf vessels. The frozenplant material was then covered with a layer of 1 ml of decompositionbuffer (1 ml CTAB buffer, 100 μl of N-laurylsarcosine buffer, 20 μl ofβ-mercaptoethanol and 10 μl of proteinase K solution, 10 mg/ml) andincubated at 60° C. for one hour with continuous shaking. The homogenateobtained was distributed into two Eppendorf vessels (2 ml) and extractedtwice by shaking with the same volume of chloroform/isoamyl alcohol(24:1). For phase separation, centrifugation was carried out at 8000×gand room temperature for 15 minutes in each case. The DNA was thenprecipitated at −70° C. for 30 minutes using ice-cold isopropanol. Theprecipitated DNA was sedimented at 4° C. and 10,000 g for 30 minutes andresuspended in 180 μl of TE buffer (Sambrook et al., 1989, Cold SpringHarbor Laboratory Press: ISBN 0-87969-309-6). For further purification,the DNA was treated with NaCl (1.2 M final concentration) andprecipitated again at −70° C. for 30 minutes using twice the volume ofabsolute ethanol. After a washing step with 70% ethanol, the DNA wasdried and subsequently taken up in 50 μl of H₂O+ RNAse (50 mg/ml finalconcentration). The DNA was dissolved overnight at 4° C. and the RNAsedigestion was subsequently carried out at 37° C. for 1 hour. Storage ofthe DNA took place at 4° C.

Example 3

Isolation of total RNA and poly-(A)+ RNA and cDNA Library Constructionfrom Physcomitrella patens

For the investigation of transcripts, both total RNA and poly-(A)⁺ RNAwere isolated. The total RNA was obtained from wild-type 9 day oldprotonemata following the GTC-method (Reski et al. 1994, Mol. Gen.Genet., 244:352-359). The Poly(A)+ RNA was isolated using Dyna Beads^(R)(Dynal, Oslo, Norway) following the instructions of the manufacturersprotocol. After determination of the concentration of the RNA or of thepoly(A)+ RNA, the RNA was precipitated by addition of 1/10 volumes of 3M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at −70° C.

For cDNA library construction, first strand synthesis was achieved usingMurine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany)and oligo-d(T)-primers, second strand synthesis by incubation with DNApolymerase I, Klenow enzyme and RNAseH digestion at 12° C. (2 hours),16° C. (1 hour) and 22° C. (1 hour). The reaction was stopped byincubation at 65° C. (10 minutes) and subsequently transferred to ice.Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche,Mannheim) at 37° C. (30 minutes). Nucleotides were removed byphenol/chloroform extraction and Sephadex G50 spin columns. EcoRIadapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends byT4-DNA-ligase (Roche, 12° C., overnight) and phosphorylated byincubation with polynucleotide kinase (Roche, 37° C., 30 minutes). Thismixture was subjected to separation on a low melting agarose gel. DNAmolecules larger than 300 base pairs were eluted from the gel, phenolextracted, concentrated on Elutip-D-columns (Schleicher and Schuell,Dassel, Germany) and were ligated to vector arms and packed into lambdaZAPII phages or lambda ZAP-Express phages using the Gigapack Gold Kit(Stratagene, Amsterdam, Netherlands) using material and following theinstructions of the manufacturer.

Example 4

Sequencing and Function Annotation of Physcomitrella patens ESTs

cDNA libraries as described in Example 3 were used for DNA sequencingaccording to standard methods, and in particular, by the chaintermination method using the ABI PRISM Big Dye Terminator CycleSequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany).Random Sequencing was carried out subsequent to preparative plasmidrecovery from cDNA libraries via iii vivo mass excision,retransformation, and subsequent plating of DH10B on agar plates(material and protocol details from Stratagene, Amsterdam, Netherlands.Plasmid DNA was prepared from overnight grown E. coli cultures grown inLuria-Broth medium containing ampicillin (see Sambrook et al. 1989 ColdSpring Harbor Laboratory Press: ISBN 0-87969-309-6) on a Qiagene DNApreparation robot (Qiagen, Hilden) according to the manufacturer'sprotocols. Sequencing primers with the following nucleotide sequenceswere used: SEQ ID NO:40 5′-CAGGAAACAGCTATGACC-3′ SEQ ID NO:415′-CTAAAGGGAACAAAAGCTG-3′ SEQ ID NO:42 5′-TGTAAAACGACGGCCAGT-3′

Sequences were processed and annotated using the software packageEST-MAX commercially provided by Bio-Max (Munich, Germany). The programincorporates practically all bioinformatics methods important forfunctional and structural characterization of protein sequences. Forreference the website at pedant.mips.biochem.mpg.de. The most importantalgorithms incorporated in EST-MAX are: FASTA: Very sensitive sequencedatabase searches with estimates of statistical significance; Pearson W.R. (1990) Rapid and sensitive sequence comparison with FASTP and FASTA.Methods Enzymol. 183:63-98; BLAST: Very sensitive sequence databasesearches with estimates of statistical significance. Altschul S. F.,Gish W., Miller W., Myers E. W., and Lipman D. J. Basic local alignmentsearch tool. Journal of Molecular Biology 215:403-10; PREDATOR:High-accuracy secondary structure prediction from single and multiplesequences. Frishman, D. and Argos, P. (1997) 75% accuracy in proteinsecondary structure prediction. Proteins, 27:329-335; CLUSTALW: Multiplesequence alignment. Thompson, J. D., Higgins, D. G. and Gibson, T. J.(1994) CLUSTAL W: improving the sensitivity of progressive multiplesequence alignment through sequence weighting, positions-specific gappenalties and weight matrix choice. Nucleic Acids Research,22:4673-4680; TMAP: Transmembrane region prediction from multiplyaligned sequences. Persson, B. and Argos, P. (1994) Prediction oftransmembrane segments in proteins utilizing multiple sequencealignments. J. Mol. Biol. 237:182-192; ALOM2: Transmembrane regionprediction from single sequences. Klein, P., Kanehisa, M., and DeLisi,C. Prediction of protein function from sequence properties: Adiscriminate analysis of a database. Biochim. Biophys. Acta 787:221-226(1984). Version 2 by Dr. K. Nakai; PROSEARCH: Detection of PROSITEprotein sequence patterns. Kolakowski L. F. Jr., Leunissen J. A. M.,Smith J. E. (1992) ProSearch: fast searching of protein sequences withregular expression patterns related to protein structure and function.Biotechniques 13, 919-921; BLIMPS. Similarity searches against adatabase of ungapped blocks. J. C. Wallace and Henikoff S., (1992);PATMAT: A searching and extraction program for sequence, pattern andblock queries and databases, CABIOS 8:249-254. Written by Bill Alford.

Example 5

Identification of Physcomitrella patens ORFs Corresponding to PK-6,PK-7, PK-8, PK-9, CK-1, CK-2, CK-3, MPK-2, MPK-3, MPK-4, MPK-5, CPK-1and CPK-2

The Physcomitrella patens partial cDNAs (ESTs) shown in Table 1 belowwere identified in the Physcomitrella patens EST sequencing programusing the program EST-MAX through BLAST analysis. The SequenceIdentification Numbers corresponding to these ESTs are as follows: PK-6(SEQ ID NO:1), PK-7 (SEQ ID NO:2), PK-8 (SEQ ID NO:3), PK-9 (SEQ IDNO:4), CK-1 (SEQ ID NO:5), CK-2 (SEQ ID NO:6), CK-3 (SEQ ID NO:7), MPK-2(SEQ ID NO:8), MPK-3 (SEQ ID NO:9), MPK-4 (SEQ ID NO:10), MPK-5 (SEQ IDNO:11), CPK-1 (SEQ ID NO:12) and CPK-2 (SEQ ID NO:13). TABLE 1Functional ORF Name categories Function Sequence code position PpPK-6Protein Kinase serine/threonine protein c_pp004044242r 1-474 kinase likeprotein PpPK-7 Protein Kinase cdc2-like protein kinase s_pp001031042f1-267 cdc2MsF PpPK-8 Protein Kinase protein kinase homologc_pp004044100r 1-581 F13C5.120 PpPK-9 Protein Kinase protein kinase;similar to c_pp004071077r 709-137  human PKX1 PpCK-1 Protein Kinasereceptor protein kinase c_pp001062017r 1160-1    PpCK-2 Protein Kinasekasein kinase c_pp004038371r 1909-1421  PpCK-3 Protein Kinase caseinkinase II catalytic c_pp004076164r 2-877 subunit PpMPK-2 Protein Kinasemitogen-activated protein c_pp004041329r 952-293  kinase 6 PpMPK-3Protein Kinase big MAP kinase 1c c_pp004061263r 221-550  PpMPK-4 ProteinKinase protein kinase MEK1 (EC c_pp001064077r 1153-596   2.7.1.—)PpMPK-5 Protein Kinase protein kinase MEK1 c_pp004064129r 114-233 PpCPK-1 Protein Kinase protein kinase c_pp004014376r 1084-173   PpCPK-2Protein Kinase calcium-dependent protein c_pp004038141r 422-1213  kinasePpPK-6 Protein Kinase cdc2-like protein kinase s_pp001031042f 1-267cdc2MsF

TABLE 2 Degree of Amino Acid Identity and Similarity of PpPK-6 and OtherHomologous Proteins (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum 62) Swiss-Prot # O81106Q9LUL4 Q9ZQZ2 Q9MAS2 Q9LK66 Protein LEUCINE- SERINE/THREONINE PUTATIVEPUTATIVE PROTEIN name RICH PROTEIN LRR LRR KINASE- REPEAT KINASE-RECEPTOR- RECEPTOR LIKE TRANSMEMBRANE LIKE LINKED PROTEIN PROTEINPROTEIN PROTEIN PROTEIN KINASE KINASE 2 KINASE Species Zea maysArabidopsis Arabidopsis Arabidopsis Arabidopsis (Maize) thalianathaliana thaliana thaliana (Mouse-ear (Mouse-ear (Mouse-ear (Mouse-earcress) cress) cress) cress) Identity % 42% 42% 38% 37% 37% Similarity %54% 52% 50% 49% 48%

TABLE 3 Degree of Amino Acid Identity and Similarity of PpPK-7 and OtherHomologous Proteins (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum 62) Swiss-Prot # P25859O49120 Q38774 P93321 Q9ZVI4 Protein CELL CYCLIN- CELL CDC2 PUTATIVE nameDIVISION DEPENDENT DIVISION KINASE SERINE/THREONINE CONTROL KINASE 1CONTROL HOMOLOG PROTEIN PROTEIN 2 PROTEIN 2 CDC2MSD KINASE HOMOLOG BHOMOLOG C Species Arabidopsis Dunaliella Antirrhinum MedicagoArabidopsis thaliana tertiolecta majus sativa thaliana (Mouse-ear(Garden (Alfalfa) (Mouse-ear cress) snapdragon) cress) Identity % 70%68% 70% 69% 69% Similarity % 79% 76% 81% 79% 77%

TABLE 4 Degree of Amino Acid Identity and Similarity of PpPK-8 and OtherHomologous Proteins (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum 62) Swiss-Prot # O82754Q9M085 Q02779 Q05609 Q39886 Protein PUTATIVE PROTEIN MITOGEN-SERINE/THREONINE- PROTEIN name SERINE/THREONINE KINASE- ACTIVATEDPROTEIN KINASE KINASE LIKE PROTEIN KINASE CTR1 PROTEIN KINASE KINASEKINASE 10 Species Arabidopsis Arabidopsis Homo sapiens ArabidopsisGlycine thaliana thaliana (Human) thaliana max (Mouse-ear (Mouse-ear(Mouse-ear (Soybean) cress) cress) cress) Identity % 25% 26% 27% 27% 26%Similarity % 42% 40% 38% 40% 40%

TABLE 5 Degree of Amino Acid Identity and Similarity of PpPK-9 and OtherHomologous Proteins (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum 62) Swiss-Prot # Q9SL77P34099 Q9TXB8 P40376 Q9SXP9 Protein PUTATIVE CAMP- SERINE/ CAMP- CAMP-name CAMP- DEPENDENT THREONINE DEPENDENT DEPENDENT DEPENDENT PROTEINPROTEIN PROTEIN PROTEIN PROTEIN KINASE KINASE KINASE KINASE KINASECATALYTIC CATALYTIC CATALYTIC SUBUNIT SUBUNIT SUBUNIT SpeciesArabidopsis Dictyostelium Dictyostelium Schizosaccharomyces Euglenathaliana discoideum pombe gracilis (Mouse-ear (Slime mold) (Fissionyeast) cress) Identity % 45% 33% 32% 33% 28% Similarity % 60% 48% 48%50% 40%

TABLE 6 Degree of Amino Acid Identity and Similarity of PpCK-1 and OtherHomologous Proteins (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum 62) Swiss-Prot # Q9SZI1Q9ZUP4 P42158 Q9LW62 Q39050 Protein COL-0 PUTATIVE CASEIN CASEIN CASEINname CASEIN CASEIN KINASE I, KINASE KINASE I KINASE I- KINASE I DELTALIKE ISOFORM PROTEIN LIKE Species Arabidopsis Arabidopsis ArabidopsisArabidopsis Arabidopsis thaliana thaliana thaliana thaliana thaliana(Mouse-ear (Mouse-ear (Mouse-ear (Mouse-ear (Mouse-ear cress) cress)cress) cress) cress) Identity % 49% 48% 48% 46% 40% Similarity % 62% 61%61% 58% 52%

TABLE 7 Degree of Amino Acid Identity and Similarity of PpCK-2 and OtherHomologous Proteins (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum 62) Swiss-Prot # Q9SZI1P42158 Q9ZWB3 Q9ZUP4 Q9LSX4 Protein COL-0 CASEIN ADK1 PUTATIVE CASEINname CASEIN KINASE I CASEIN KINASE I KINASE I- KINASE I LIKE PROTEINSpecies Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsisthaliana thaliana thaliana thaliana thaliana (Mouse-ear (Mouse-ear(Mouse-ear (Mouse-ear (Mouse-ear cress) cress) cress) cress) cress)Identity % 64% 59% 60% 58% 57% Similarity % 73% 66% 72% 67% 69%

TABLE 8 Degree of Amino Acid Identity and Similarity of PpCK-3 and OtherHomologous Proteins (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum 62) Swiss-Prot # O64816Q9ZR52 P28523 Q9SN18 Q08466 Protein PUTATIVE CASEIN CASEIN CASEIN CASEINname CASEIN KINASE II KINASE II, KINASE II, KINASE II, KINASE II ALPHAALPHA ALPHA ALPHA CATALYTIC SUBUNIT CHAIN CHAIN 2 CHAIN 2 SUBUNIT (CKII) Species Arabidopsis Zea mays Zea mays Arabidopsis Arabidopsisthaliana (Maize) (Maize) thaliana thaliana (Mouse-ear (Mouse-ear(Mouse-ear cress) cress) cress) Identity % 87% 89% 89% 88% 88%Similarity % 93% 94% 93% 93% 93%

TABLE 9 Degree of Amino Acid Identity and Similarity of PpMPK-2 andOther Homologous Proteins (Pairwise Comparison was used: gap penalty:10; gap extension penalty: 0.1; score matrix: blosum 62) Swiss-Prot #Q9M136 Q40531 Q39024 Q40353 Q07176 Protein MAP MITOGEN- MITOGEN-MITOGEN- MITOGEN- name KINASE 4 ACTIVATED ACTIVATED ACTIVATED ACTIVATEDPROTEIN PROTEIN PROTEIN PROTEIN KINASE KINASE KINASE KINASE HOMOLOGHOMOLOG 4 HOMOLOG HOMOLOG NTF6 MMK2 MMK1 Species Arabidopsis NicotianaArabidopsis Medicago Medicago thaliana tabacum thaliana sativa sativa(Mouse-ear (Common (Mouse-ear (Alfalfa) (Alfalfa) cress) tobacco) cress)Identity % 70% 69% 69% 68% 66% Similarity % 80% 78% 80% 79% 76%

TABLE 10 Degree of Amino Acid Identity and Similarity of PpMPK-3 andOther Homologous Proteins (Pairwise Comparison was used: gap penalty:10; gap extension penalty: 0.1; score matrix: blosum 62) Swiss-Prot #Q9SUX2 P13983 Q41192 O70495 Q9RLD9 Protein EXTENSIN- EXTENSIN NAPRP3PLENTY- FERULOYL- name LIKE OF- COA PROTEIN PROLINES- SYNTHETASE 101Species Arabidopsis Nicotiana Nicotiana Mus Pseudomonas thaliana tabacumalata musculus sp. (Mouse-ear (Common (Winged (Mouse) cress) tobacco)tobacco) (Persian tobacco) Identity % 12% 15% 22% 18% 11% Similarity %21% 22% 30% 26% 20%

TABLE 11 Degree of Amino Acid Identity and Similarity of PpMPK-4 andOther Homologous Proteins (Pairwise Comparison was used: gap penalty:10; gap extension penalty: 0.1; score matrix: blosum 62) Swiss-Prot #O49975 O48616 Q9M6Q9 O80395 Q9S7U9 Protein PROTEIN MAP KINASE MAP KINASEMAP KINASE MAP2K name KINASE KINASE KINASE KINASE 2 BETA ZMMEK1 PROTEINSpecies Zea mays Lycopersicon Nicotiana Arabidopsis Arabidopsis (Maize)esculentum tabacum thaliana thaliana (Tomato) (Common (Mouse-ear(Mouse-ear tobacco) cress) cress) Identity % 59% 54% 53% 50% 50%

TABLE 12 Degree of Amino Acid Identity and Similarity of PpMPK-5 andOther Homologous Proteins (Pairwise Comparison was used: gap penalty:10; gap extension penalty: 0.1; score matrix: blosum 62) Swiss-Prot #O49975 O48616 Q9M6Q9 O80395 Q9S7U9 Protein PROTEIN MAP MAP MAP MAP2KBETA name KINASE KINASE KINASE KINASE PROTEIN ZMMEK1 KINASE KINASEKINASE 2 Species Zea mays Lycopersicon Nicotiana Arabidopsis Arabidopsis(Maize) esculentum tabacum thaliana thaliana (Tomato) (Common (Mouse-ear(Mouse-ear tobacco) cress) cress) Identity % 59% 54% 53% 50% 50%Similarity % 72% 66% 66% 62% 62%

TABLE 13 Degree of Amino Acid Identity and Similarity of PpCPK-1 andOther Homologous Proteins (Pairwise Comparison was used: gap penalty:10; gap extension penalty: 0.1; score matrix: blosum 62) Swiss-Prot #Q9SCS2 O04290 P53681 P93520 Q41792 Protein CDPK- CDPK- CDPK- CALCIUM/CALCDPK- name RELATED RELATED RELATED MODULIN- RELATED PROTEIN PROTEINPROTEIN DEPENDENT PROTEIN KINASE KINASE KINASE PROTEIN KINASE KINASEHOMOLOG Species Arabidopsis Arabidopsis Daucus Zea mays Zea maysthaliana thaliana carota (Maize) (Maize) (Mouse-ear (Mouse-ear (Carrot)cress) cress) Identity % 64% 64% 63% 63% 63% Similarity % 76% 76% 75%73% 74%

TABLE 14 Degree of Amino Acid Identity and Similarity of PpCPK-2 andOther Homologous Proteins (Pairwise Comparison was used: gap penalty:10; gap extension penalty: 0.1; score matrix: blosum 62) Swiss-Prot #Q9S7Z4 Q42479 Q41790 O81390 Q9ZPM0 Protein CALCIUM- CALCIUM- CALCIUM-CALCIUM- CA2+- name DEPENDENT DEPENDENT DEPENDENT DEPENDENT DEPENDENTPROTEIN PROTEIN PROTEIN PROTEIN PROTEIN KINASE KINASE KINASE KINASEKINASE Species Marchantia Arabidopsis Zea mays Nicotiana Mesembryantpolymorpha thaliana (Maize) tabacum hemum (Liverwort) (Mouse-ear (Commoncrystallinum cress) tobacco) (Common ice plant) Identity % 66% 62% 59%59% 59% Similarity % 75% 73% 70% 68% 70%

TABLE 14-1 Degree of Amino Acid Identity and Similarity of PpCK2-1 andOther Proteins (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum 62) Public Database #S31099 NP_919109 Q9ZR52 Q94IG2 Q70Z24 Polypeptide Casein kinase IICasein kinase II Casein kinase II Casein kinase II Protein kinase namealpha subunit alpha subunit alpha CK2 alpha chain Species ArabidopsisOryza sativa Zea mays Triticum Nicotiana thaliana aestivum tabacumIdentity % 86.2 88.9 88.3 88.0 88.3 Similarity % 92.5 94.6 94.3 93.493.4

Example 6

Cloning of the Full-Length Physcomitrella patens cDNA Encoding for PK-6,PK-7, PK-8, PK-9, CK-1, CK-2, CK-3, MPK-2, MPK-3, MPK-4, MPK-5, CPK-1and CPK-2

To isolate the clones encoding PK-6 (SEQ ID NO:14), PK-7 (SEQ ID NO:15),PK-8 (SEQ ID NO:16), PK-9 (SEQ ID NO:17), CK-1 (SEQ ID NO:18), CK-2 (SEQID NO:19), CK-3 (SEQ ID NO:20), CK2-1 (SEQ ID NO:129), MPK-2 (SEQ IDNO:21), MPK-3 (SEQ ID NO:22), MPK-4 (SEQ ID NO:23), MPK-5 (SEQ IDNO:24), CPK-1 (SEQ ID NO:25) and CPK-2 (SEQ ID NO:26) fromPhyscomitrella patens, cDNA libraries were created with SMART RACE cDNAAmplification kit (Clontech Laboratories) following manufacturer'sinstructions. Total RNA isolated as described in Example 3 was used asthe template. The cultures were treated prior to RNA isolation asfollows: Salt Stress: 2, 6, 12, 24, 48 hours with 1-M NaCl-supplementedmedium; Cold Stress: 4° C. for the same time points as for salt; DroughtStress: cultures were incubated on dry filter paper for the same timepoints as for salt.

5′ RACE Protocol

The EST sequences PK-6 (SEQ ID NO:1), PK-7 (SEQ ID NO:2), PK-8 (SEQ IDNO:3), PK-9 (SEQ ID NO:4), CK-1 (SEQ ID NO:5), CK-2 (SEQ ID NO:6), CK-3(SEQ ID NO:7), CK2-1 (SEQ ID NO:129), MPK-2 (SEQ ID NO:8), MPK-3 (SEQ IDNO:9), MPK-4 (SEQ ID NO:10), MPK-5 (SEQ ID NO:11), CPK-1 (SEQ ID NO:12)and CPK-2 (SEQ ID NO:13) identified from the database search asdescribed in Example 4 were used to design oligos for RACE (see Table15). The extended sequences for these genes were obtained by performingRapid Amplification of cDNA Ends polymerase chain reaction (RACE PCR)using the Advantage 2 PCR kit (Clontech Laboratories) and the SMART RACEcDNA amplification kit (Clontech Laboratories) using a Biometra T3Thermocycler following the manufacturer's instructions. The sequencesobtained from the RACE reactions corresponded to full-length codingregions of CC-2 and CC-3 and were used to design oligos for full-lengthcloning of the respective genes (see below full-length amplification).

Full-Length Amplification

Full-length clones corresponding PK-6 (SEQ ID NO:14), PK-7 (SEQ IDNO:15), PK-8 (SEQ ID NO:16), PK-9 (SEQ ID NO:17), CK-1 (SEQ ID NO:18),CK-2 (SEQ ID NO:19), CK-3 (SEQ ID NO:20), CK2-1 (SEQ ID NO:129), MPK-2(SEQ ID NO:21), MPK-3 (SEQ ID NO:22), MPK-4 (SEQ ID NO:23), MPK-5 (SEQID NO:24), CPK-1 (SEQ ID NO:25) and CPK-2 (SEQ ID NO:26) were obtainedby performing polymerase chain reaction (PCR) with gene-specific primers(see Table 15) and the original EST as the template. The conditions forthe reaction were standard conditions with PWO DNA polymerase (Roche).PCR was performed according to standard conditions and to manufacturer'sprotocols (Sambrook et al., 1989 Molecular Cloning, A Laboratory Manual.2nd Edition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor,N.Y., Biometra T3 Thermocycler). The parameters for the reaction were:five minutes at 94° C. followed by five cycles of one minute at 94° C.,one minute at 50° C. and 1.5 minutes at 72° C. This was followed bytwenty five cycles of one minute at 94° C., one minute at 65° C. and 1.5minutes at 72° C.

The amplified fragments were extracted from agarose gel with a QIAquickGel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector(Invitrogen) following manufacturer's instructions. Recombinant vectorswere transformed into Top10 cells (Invitrogen) using standard conditions(Sambrook et al. 1989. Molecular Cloning, A Laboratory Manual. 2ndEdition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.).Transformed cells were selected for on LB agar containing 100 μg/mlcarbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside)and 0.8 mg IPTG (isopropylthio-β-D-galactoside) grown overnight at 37°C. White colonies were selected and used to inoculate 3 ml of liquid LBcontaining 100 μg/ml ampicillin and grown overnight at 37° C. PlasmidDNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) followingmanufacturer's instructions. Analyses of subsequent clones andrestriction mapping was performed according to standard molecularbiology techniques (Sambrook et al., 1989 Molecular Cloning, ALaboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press.Cold Spring Harbor, N.Y.). TABLE 15 Scheme and primers used for cloningof full-length clones Final product Isolation Gene Sites Method PrimersRace Primers RT-PCR PpPK-6 XmaI/HpaI 5′ RACE and RC782: RC858: RT-PCRfor (SEQ ID NO:43) (SEQ ID NO:46) Full-length CCACGGTCTTCGGATCCCGGGTGAGT clone CTGCTGGTCGTG ATCACTTACGGTG RC783: CGA (SEQ ID NO:44)RC859: GCAGCAGAGCAC (SEQ ID NO:47) CACCAGCGGCTAT GCGTTAACTCGAC (SEQ IDNO:45) CAAGGTCACTATT GCGCCCAGTGAG CCAAGCA TAGCTCCAGCATT PpPK-7 XmaI/HpaI5′ RACE and RC250: RC590: RT-PCR for (SEQ ID NO:48) (SEQ ID NO:49)Full-length CGGTGGCCACCTC ATCCCGGGAGTGG clone GTTCCTGTGGTT GTGGTTGGACTGTAAGGA RC591: (SEQ ID NO:50) GCGTTAACCTTCG TCTTGGACAGGTA GAGGTTAC PpPK-8XmaI/HpaI 5′ RACE and (SEQ ID NO:51) RC1016: RT-PCR for GACTCAGCCCCGT(SEQ ID NO:52) FuIl-length AATCCTTCAACA ATCCCGGGCAACG cloneAGAAGCATTCGAG ATGGC RC1021: (SEQ ID NO:53) GCGTTAACGAGCA TCACGATACTCGGTGATTTC PpPK-9 XmaI/SacI 5′ RACE and RC263: RC831: RT-PCR for (SEQ IDNO:54) (SEQ ID NO:55) Full-length CGACGGCTAATA ATCCCGGGCTGTG cloneCCACGTTGGCGAC ATGTCGGTGTGGT CA GCTCTGC RC832: (SEQ ID NO:56)GCGAGCTCGCACC ACTGAATGATGGA GACTCAGG PpCK-1 XmaI/HpaI 5′ RACE and (SEQID NO:57) RC614: RT-PCR for CGACCGCAGCCC (SEQ ID NO:58) Full-lengthATGAGGAAGTTA ATCCCGGGCTCAC clone T GTAGTGCACTGAA TGTGTC RC615: (SEQ IDNO:59) GCGTTAACATGCC CATCTTCTCATACT CAGACC PpCK-2 XmaI/HpaI 5′ RACE and(SEQ ID NO:60) RC1012: RT-PCR for CTCGCCTACCAA (SEQ ID NO:61)Full-length GCCCCATTAGAA ATCCCGGGTTGTC clone A GAGGACGGAGAG AGAAGAGRC1015: (SEQ ID NO:62) GCGTTAACCTTAG GAATCGTATGGCA GAGAGCT PpCK-3HpaI/SacI 5′ RACE and (SEQ ID NO:63) C640: RT-PCR for GCTTCACAATGT (SEQID NO:64) Full-length TGGGCCCTCCAC GCGTTAACGGGAG clone A GAAGGTCGGGGGAAGAGACG RC641: (SEQ ID NO:65) GCGAGCTCAGCGC TTCGCACAACTGA GAAACCTPpMPK-2 XmaI/HpaI 5′ RACE and (SEQ ID NO:66) RC664: RT-PCR forACGAGAAGGTTG (SEQ ID NO:67) Full-length GTGGGCTTCAAG ATCCCGGGCGAGC cloneT CATGGCGCCACTT GCTT RC665: (SEQ ID NO:68) GCGTTAACGCCGA GCAACAATGTCTGCTGGATG PpMPK-3 XmaI/SacI 5′ RACE and RC268: RC662: RT-PCR for (SEQ IDNO:69) (SEQ ID NO:70) Full-length CCCGGGTAAGCCAT ATCCCGGGCTTGT cloneCGGAGTGTGGAA ATTGGCTCGGATA ATTT RC663: (SEQ ID NO:71) GCGTTAACGGCAATATCTGCACAGCC GTTCACT PpMPK-4 XmaI/SacI 5′ RACE and (SEQ ID NO:72)RC1001: RT-PCR for GTGTCTCGCTGG (SEQ ID NO:73) Full-length GCCAAGGAATGAATCCCGGGCGGTC clone A GAGTCGTATTAGG TGTTGTTTC RC1005: (SEQ ID NO:74)GAGCTCCGGTAGG TCCGACCTGTTCA ATTG PpMPK-5 XmaI/SacI 5′ RACE and RC266:RC572: RT-PCR for (SEQ ID NO:75) (SEQ ID NO:76) Full-length GACGACGCGAAGATCCCGGGAGAGG clone CCCGGTGTGGTTG CTGATCTGATGCT A ACAGT RC573: (SEQ IDNO:77) ATGAGCTCTGGCG GATTGGCGAGGTA GTTCGAC PpCPK-1 XmaI/HpaI 5′ RACE andRC526: RC817: RT-PCR for (SEQ ID NO:78) (SEQ ID NO:82) Full-lengthCGGCGCAACGTA ATCCCGGGTGTAG clone GTATGCGCTTCCA GCGGGCGAGGTTC GATGCRC723N: RC818: (SEQ ID NO:79) (SEQ ID NO:83) CGCGGTGAACAA GCGTTAACGACAACACCTTGCAGGTG CCGGAGTAGAACG AC GCAGTCCA RC767: (SEQ ID NO:80)GCTCGGGTCAGCC CTCAACACCGCA (SEQ ID NO:81) GTTAAAGCTTGTG CAGCAGTCATGCPpCPK-2 XmaI/HpaI 5′ RACE and (SEQ ID NO:84) RC703: RT-PCR forAGAAGCGAGGA (SEQ ID NO:85) Full-length ATGGGCAGGGAC ATCCCGGGCGAAC cloneGA TGCGATCTGAGAT TCCAAC RC704: (SEQ ID NO:86) GCGTTAACGAGATCCAACCGAAGCCA TCCTACGA PpCK2-1 EcoRV/ 5′ RACE and RC296: RC648: EcoRVRT-PCR for (SEQ ID NO:131) (SEQ ID NO:87) Full-length CACCCGGGCCTTGCGATATCGGCG clone GGACATCGCTCC CAGAACATTGGA AG AAGTCGGTT RC649: (SEQ IDNO:88) GCGATATCGCCTG CGCGTGTTGAATA TGGAAGA

Example 7

Engineering Stress-Tolerant Arabidopsis Plants by Over-Expressing theGenes PK-6, PK-7, PK-8, PK-9, CK-1, CK-2, CK-3, MPK-2, MPK-3, MPK-4,MPK-5, CPK-1 and CPK-2

Cloning of PK-6, PK-7, PK-8, PK-9, CK-1, CK-2, CK-3, MPK-2, MPK-3,MPK-4, MPK-5, CPK-1 and CPK-2 into a Binary vector

The fragments containing the different Physcomitrella patens proteinkinases were subcloned from the recombinant PCR2.1 TOPO vectors bydouble digestion with restriction enzymes (see Table 16) according tomanufacturer's instructions. The subsequence fragment was excised fromagarose gel with a QIAquick Gel Extraction Kit (QIAgen) according tomanufacture's instructions and ligated into a binary vector containing aselectable marker gene. The resulting recombinant vector contained thecorresponding PKSRP gene in the sense orientation under the constitutivesuper promoter. TABLE 16 Listed are the names of the various constructsof the Physcomitrella patens transcription factors used for planttransformation Enzymes used to generate gene Enzymes used to BinaryVector Gene fragment restrict pBPSJH001 Construct PpPK-6 XmaI/HpaIXmaI/SacI pBPSJyw022 PpPK-7 XmaI/HpaI XmaI/Ecl136 pBPSJyw012 PpPK-8XmaI/HpaI XmaI/Ecl136 pBPSJYW030 PpPK-9 XmaI/SacI XmaI/SacI PBPSERG010PpCK-1 XmaI/HpaI XmaI/Ecl136 pBPSSY012 PpCK-2 XmaI/HpaI XmaI/Ecl136pBPSJyw034 PpCK-3 HpaI/SacI SmaI/SacI pBPSSY011 PpMPK-2 XmaI/HpaIXmaI/Ecl136 pBPSSY016 PpMPK-3 XmaI/HpaI XmaI/Ecl136 pBPSJyw014 PpMPK-4XmaI/SacI XmaI/SacI pBPSJyw025 PpMPK-5 XmaI/SacI XmaI/SacI PBPSERG009PpCPK-1 XmaI/HpaI XmaI/Ecl136 PBPSERG019 PpCPK-2 XmaI/HpaI XmaI/Ecl136pBPSJyw008Agrobacterium Transformation

The recombinant vectors were transformed into Agrobacterium tumefaciensC58C1 and PMP90 according to standard conditions (Hoefgen andWillmitzer, 1990).

Plant Transformation

Arabidopsis thaliana ecotype C24 were grown and transformed according tostandard conditions (Bechtold 1993, Acad. Sci. Paris. 316:1194-1199;Bent et al. 1994, Science 265:1856-1860).

Screening of Transformed Plants

T1 seeds were sterilized according to standard protocols (Xiong et al.1999, Plant Molecular Biology Reporter 17: 159-170). Seeds were platedon ½ Murashige and Skoog media (MS) (Sigma-Aldrich) pH 5.7 with KOH,0.6% agar and supplemented with 1% sucrose, 0.5 g/L2-[N-Morpholino]ethansulfonic acid (MES) (Sigma-Aldrich), 50 μg/mlselection agent, 500 μg/ml carbenicillan (Sigma-Aldrich) and 2 μg/mlbenomyl (Sigma-Aldrich). Seeds on plates were vernalized for four daysat 4° C. The seeds were germinated in a climatic chamber at an airtemperature of 22° C. and light intensity of 40 micromols^(−1m2) (whitelight; Philips TL 65W/25 fluorescent tube) and 16 hours light and 8hours dark day length cycle. Transformed seedlings were selected after14 days and transferred to ½ MS media pH 5.7 with KOH 0.6% agar platessupplemented with 0.6% agar, 1% sucrose, 0.5 g/L MES (Sigma-Aldrich),and 2 μg/ml benomyl (Sigma-Aldrich) and allowed to recover forfive-seven days.

Growth Screen Under Water-Limited Conditions

The PpCK2-1 or PpCK-3 gene was overexpressed in A. thaliana under thecontrol of a constitutive promoter. T2 and/or T3 seeds were screened forresistance to the selection agent conferred by the selectable markergene on plates, and positive plants were transplanted into soil andgrown in a growth chamber for 3 weeks. Soil moisture was maintainedthroughout this time at approximately 50% of the maximum water-holdingcapacity of soil.

The total water lost (transpiration) by the plant during this time wasmeasured. After 3 weeks, the entire above-ground plant material wascollected, dried at 65° C. for 2 days and weighed. The results are shownin Tables 16-1 and 16-2. The ratio of above-ground plant dry weight (DW)to plant water use is Water Use Efficiency (WUE). Tables 16-1 and 16-2present WUE and DW, respectively, for independent transformation events(lines). Least square means, standard errors and significant value (p)of a line compared to wild-type controls from an Analysis of Varianceare presented. The percent improvement from wild-type control plants forWUE (Table 16-1) and DW (Table 16-2) for both PpCK2-1 (EST 391) andPpCK-3 (EST 293) overexpressing plants are also presented. TABLE 16-1Least Meas- Square Standard % urement Genotype Line Mean ErrorImprovement P WUE Wild-type 2.270 0.081 PpCK-3 8 2.444 0.166 8 0.349PpCK-3 7 2.761 0.204 22 0.0272 Wild-type 1.446 0.103 PpCK2-1 8 1.5770.241 9 0.618

TABLE 16-2 Least Meas- Square Standard % urement Genotype Line MeanError Improvement P DW Wild-type 0.136 0.011 PpCK-3 8 0.245 0.023 81<.0001 PpCK-3 7 0.275 0.028 103 <.0001 Wild-type 0.088 0.009 PpCK2-1 80.144 0.021 64 0.015Drought Tolerance Screening

T1 seedlings were transferred to dry, sterile filter paper in a petridish and allowed to desiccate for two hours at 80% RH (relativehumidity) in a Percieval Growth Cabinet MLR-350H, micromols^(−1m2)(white light; Philips TL 65W/25 fluorescent tube). The RH was thendecreased to 60% and the seedlings were desiccated further for eighthours. Seedlings were then removed and placed on ½ MS 0.6% agar platessupplemented with 2 μg/ml benomyl (Sigma-Aldrich) and 0.5 g/L MES(Sigma-Aldrich) and scored after five days.

Under drought stress conditions, PpPK-6 over-expressing Arabidopsisthaliana plants showed a 95% (20 survivors from 21 stressed plants)survival rate to the stress screening; PpPK-8, 40% (2 survivors from 5stressed plants), PpPK-9, 78% (38 survivors from 49 stressed plants),PpCK-1, 50% (5 survivors from 10 stressed plants), PpCK-2, 52% (16survivors from 31 stressed plants), PpCK-3, 60% (3 survivors from 5stressed plants), PpMPK-2, 100% (52 survivors from 52 stressed plants),PpMPK-3, 98% (44 survivors from 45 stressed plants), PpMPK-4, 92% (11survivors from 12 stressed plants), PpMPK-5, 100% (9 survivors from 9stressed plants), PpCPK-1, 60% (12 survivors from 20 stressed plants),PpCPK-2, 89% (17 survivors from 19 stressed plants), whereas theuntransformed control only showed a 11% survival rate (1 survivor from 9stressed plants). It is noteworthy that the analyses of these transgeniclines were performed with T1 plants, and therefore, the results will bebetter when a homozygous, strong expresser is found. TABLE 17 Summary ofthe drought stress tests Drought Stress Test Gene Number of Total numberof Percentage of Name survivors plants survivors PpPK-6 20 21 95% PpPK-82 5 40% PpPK-9 38 49 78% PpCK-1 5 10 50% PpCK-2 16 31 52% PpCK-3 3 5 60%PpMPK-2 52 52 100%  PpMPK-3 44 45 98% PpMPK-4 11 12 92% PpMPK-5 9 9100% Freezing Tolerance Screening

Seedlings were moved to petri dishes containing ½ MS 0.6% agarsupplemented with 2% sucrose and 2 μg/ml benomyl. After four days, theseedlings were incubated at 4° C. for 1 hour and then covered withshaved ice. The seedlings were then placed in an EnvironmentalSpecialist ES2000 Environmental Chamber and incubated for 3.5 hoursbeginning at −1.0° C. decreasing −1° C. hour. The seedlings were thenincubated at −5.0° C. for 24 hours and then allowed to thaw at 5° C. for12 hours. The water was poured off and the seedlings were scored after 5days.

Under freezing stress conditions, PpPK-7 over-expressing Arabidopsisthaliana plants showed a 73% (8 survivors from 11 stressed plants)survival rate to the stress screening; PpPK-9, 100% (45 survivors from45 stressed plants), PpCK-1, 100% (14 survivors from 14 stressedplants), PpMPK-2, 68% (36 survivors from 53 stressed plants), PpMPK-3,92% (24 survivors from 26 stressed plants), PpCPK-2, 64% (7 survivorsfrom 11 stressed plants), whereas the untransformed control only showeda 2% survival rate (1 survivor from 48 stressed plants). It isnoteworthy that the analyses of these transgenic lines were performedwith T1 plants, and therefore, the results will be better when ahomozygous, strong expresser is found. TABLE 18 Summary of the freezingstress tests Freezing Stress Test Number Total number of Percentage ofGene Name of survivors plants survivors PpPK-7 8 11 73% PpPK-9 45 45100%  PpCK-1 14 14 100%  PpMPK-2 36 53 68% PpMPK-3 24 26 92% PpCPK-2 711 64% Control 1 48  2%Salt Tolerance Screening

Seedlings were transferred to filter paper soaked in ½ MS and placed on½ MS 0.6% agar supplemented with 2 μg/ml benomyl the night before thesalt tolerance screening. For the salt tolerance screening, the filterpaper with the seedlings was moved to stacks of sterile filter paper,soaked in 50 mM NaCl, in a petri dish. After two hours, the filter paperwith the seedlings was moved to stacks of sterile filter paper, soakedwith 200 mM NaCl, in a petri dish. After two hours, the filter paperwith the seedlings was moved to stacks of sterile filter paper, soakedin 600mM NaCl, in a petri dish. After 10 hours, the seedlings were movedto petri dishes containing ½ MS 0.6% agar supplemented with 2 μg/mlbenomyl. The seedlings were scored after 5 days.

The transgenic plants are screened for their improved salt tolerancedemonstrating that transgene expression confers salt tolerance.

Example 8

Detection of the PK-6, PK-7, PK-8, PK-9, CK-1, CK-2, CK-3, MPK-2, MPK-3,MPK-4, MPK-5, CPK-1 and CPK-2 Transgenes in the Transgenic ArabidopisLines

One leaf from a wild type and a transgenic Arabidopsis plant washomogenized in 250 μl Hexadecyltrimethyl ammonium bromide (CTAB) buffer(2% CTAB, 1.4 M NaCl, 8 mM EDTA and 20 mM Tris pH 8.0) and 1 μlβ-mercaptoethanol. The samples were incubated at 60-65° C. for 30minutes and 250 μl of Chloroform was then added to each sample. Thesamples were vortexed for 3 minutes and centrifuged for 5 minutes at18,000×g. The supernatant was taken from each sample and 150 μlisopropanol was added. The samples were incubated at room temperaturefor 15 minutes, and centrifuged for 10 minutes at 18,000×g. Each pelletwas washed with 70% ethanol, dried, and resuspended in 20 μl TE. 4 μl ofabove suspension was used in a 20 μl PCR reaction using Taq DNApolymerase (Roche Molecular Biochemicals) according to themanufacturer's instructions.

Binary vector plasmid with each gene cloned in was used as positivecontrol, and the wild-type C24 genomic DNA was used as negative controlin the PCR reactions. 10 μl PCR reaction was analyzed on 0.8%agarose—ethidium bromide gel.

PpPk-6: The primers used in the reactions are: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:90)GCGTTAACTCGACCAAGGTCACTATTCCAAGCA

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 2.8 kb fragment was produced from the positive control and thetransgenic plants.

PpPk-7: The primers used in the reactions are: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:91)GCGTTAACCTTCGTCTTGGACAGGTAGAGGTTAC

The primers were used in the first round of reactions with the followingprogram: 30 cycles of 1 minute at 94° C., 1 minute at 62° C. and 4minutes at 72° C., followed by 10 minutes at 72° C. A 1.1 kb fragmentwas generated from the positive control and the T1 transgenic plants.

PpPK-8: The primers used in the reactions were: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:92)GCGTTAACGAGCATCACGATACTCGGTGATTTC

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 1.6 kb fragment was produced from the positive control and thetransgenic plants.

PpPK-9: The primers used in the reactions are: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:93)GCGAGCTCGCACCACTGAATGATGGAGACTCAGG

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 1.4 kb fragment was produced from the positive control and thetransgenic plants.

PpCK-1: The primers used in the reactions are: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:94)GCGTTAACATGCCCATCTTCTCATACTCAGACC

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 1.7 kb fragment was produced from the positive control and thetransgenic plants.

PpCK-2: The primers used in the reactions are: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:95)GCGTTAACCTTAGGAATCGTATGGCAGAGAGCT

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 1.9 kb fragment was produced from the positive control and thetransgenic plants.

PpCK-3: The primers used in the reactions are: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:96)GCGAGCTCAGCGCTTCGCACAACTGAGAAACCT

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 1.2 kb fragment was produced from the positive control and thetransgenic plants.

PpMPK-2: The primers used in the reactions are: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:97)GCGTTAACGGCAATATCTGCACAGCCGTTCACT

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 1.7 kb fragment was produced from the positive control and thetransgenic plants.

PpMPK-3: The primers used in the reactions are: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:98)GCGTTAACGGCAATATCTGCACAGCCGTTCACT

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 2.2 kb fragment was produced from the positive control and thetransgenic plants.

PpMPK4: The primers used in the reactions are: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:99) GAGCTCCGGTAGGTCCGACCTCTTCAATTG

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 1.7 kb fragment was produced from the positive control and thetransgenic plants.

PpMPK-5: The primers used in the reactions are: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:100)ATGAGCTCTGGCGGATTGGCGAGGTAGTTCGAC

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 1.4 kb fragment was produced from the positive control and thetransgenic plants.

PpCPK-1: The primers used in the reactions are: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:101)GCGTTAACGACAACCGGAGTAGAACGGCAGTCCA

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 2.3 kb fragment was produced from the positive control and thetransgenic plants.

PpCPK-2: The primers used in the reactions are: (SEQ ID NO:89)GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO:102)GCGTTAACGAGATCCAACCGAAGCCATCCTACGA

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 2.2 kb fragment was produced from the positive control and thetransgenic plants.

The transgenes were successfully amplified from the T1 transgenic lines,but not from the wild type C24. This result indicates that the T1transgenic plants contain at least one copy of the transgenes. There wasno indication of existence of either identical or very similar genes inthe untransformed Arabidopsis thaliana control which could be amplifiedby this method.

Example 9

Detection of the PK-6, PK-7, PK-8, PK-9, CK-1, CK-2, CK-3, MPK-2, MPK-3,MPK-4, MPK-5, CPK-1 and CPK-2 Transgene mRNA in Transgenic ArabidopsisLines

Transgene expression was detected using RT-PCR. Total RNA was isolatedfrom stress-treated plants using a procedure adapted from (Verwoerd etal., 1989 NAR 17:2362). Leaf samples (50-100 mg) were collected andground to a fine powder in liquid nitrogen. Ground tissue wasresuspended in 500 μl of a 80° C., 1:1 mixture, of phenol to extractionbuffer (100 mM LiCl, 100 mM Tris pH8, 10 mM EDTA, 1% SDS), followed bybrief vortexing to mix. After the addition of 250 μl of chloroform, eachsample was vortexed briefly. Samples were then centrifuged for 5 minutesat 12,000×g. The upper aqueous phase was removed to a fresh eppendorftube. RNA was precipitated by adding 1/10^(th) volume 3M sodium acetateand 2 volumes 95% ethanol. Samples were mixed by inversion and placed onice for 30 minutes. RNA was pelleted by centrifugation at 12,000×g for10 minutes. The supernatant was removed and pellets briefly air-dried.RNA sample pellets were resuspended in 10 μl DEPC treated water. Toremove contaminating DNA from the samples, each was treated withRNase-free DNase (Roche) according to the manufacturer'srecommendations. cDNA was synthesized from total RNA using the 1^(st)Strand cDNA synthesis kit (Boehringer Mannheim) following manufacturer'srecommendations.

PCR amplification of a gene-specific fragment from the synthesized cDNAwas performed using Taq DNA polymerase (Roche) and gene-specific primers(see Table 15 for primers) in the following reaction: 1× PCR buffer, 1.5mM MgCl₂, 0.2 μM each primer, 0.2 μM dNTPs, 1 unit polymerase, 5 μl cDNAfrom synthesis reaction. Amplification was performed under the followingconditions: Denaturation, 95° C., 1 minute; annealing, 62° C., 30seconds; extension, 72° C., 1 minute, 35 cycles; extension, 72° C., 5minutes; hold, 4° C., forever. PCR products were run on a 1% agarosegel, stained with ethidium bromide, and visualized under UV light usingthe Quantity-One gel documentation system (Bio-Rad).

Expression of the transgenes was detected in the T1 transgenic line.This result indicated that the transgenes are expressed in thetransgenic lines and strongly suggested that their gene product improvedplant stress tolerance in the transgenic line. On the other hand, noexpression of identical or very similar endogenous genes could bedetected by this method. These results are in agreement with the datafrom Example 7. This greatly supports our statement that the observedstress tolerance is due to the introduced transgene. PpPK-6 (SEQ IDNO:103) CCCAGTAATAGCAGGGTTGGAGGAA (SEQ ID NO:104)GGCTGCCTGAAGATCCGCTACAGAG PpPK-7 (SEQ ID NO:105)CGTCAGGCTACTTTGCGTGGAGCAC (SEQ ID NO:106) CGGTGCTGGCTAACACCAGGCCAGAPpPK-8 (SEQ ID NO:107) ATCCCGGGCAACGAGAAGCATTCGAGATGGC (SEQ ID NO:108)GCGTTAACGAGCATCACGATACTCGGTGATTTC PpPK-9 (SEQ ID NO:109)CGTGGCATCTCTCCCGATGTTCTTA (SEQ ID NO:110) GGCCAACTGAAGGCGTGTCATGATCPpCK-1 (SEQ ID NO:111) CTCGAGGGCTCGTTCACCGTGACCT (SEQ ID NO:112)CGGAGGTAACAGTAGTCAGGCTGCTC PpCK-2 (SEQ ID NO:113)CCGCGACCCTTCCACGCATCAGCAT (SEQ ID NO:114) CCTCCAGGAAGCCTGCGCCGAGAAGPpCK-3 (SEQ ID NO:115) GGACATTGTCCGTGATCAGCAATCGA (SEQ ID NO:116)CAGCCTCTGGAACAACCAGACGCTG PpMPK-2 (SEQ ID NO:117)GTCACCGCGAGGTACAAGCCACCAC (SEQ ID NO:118) GCAGCTCTGGAGCTCTGTACCACCTPpMPK-3 (SEQ ID NO:119) ACGGCCACGTCGAGAATCTGAGCAA (SEQ ID NO:120)CGAAGTGCTCGCAAGCAATGCCGAA PpMPK-4 (SEQ ID NO:121)ATCCCGGGCGGTCGAGTCGTATTAGGTGTTGTTTC (SEQ ID NO:122)GAGCTCCGGTAGGTCCGACCTCTTCAATTG PpMPK-5 (SEQ ID NO:123)GGGCAACTGTCAATAGCAGACCTGGA (SEQ ID NO:124) GCAAGTCCCAACGAACGTGTCTCGCTPpCPK-1 (SEQ ID NO:125) GCGAAGATGACGACTGCTATTGCGA (SEQ ID NO:126)CGTGATGACTCCAATGCTCCATACG PpCPK-2 (SEQ ID NO:127)GCCAGCATCGAGGTCAGTATCCGGTGT (SEQ ID NO:128) GTCTGTGGCCTTCAGAGGCGCATCCTC

Amplification was performed under the following conditions:Denaturation, 95° C., 1 minute; annealing, 62° C., 30 seconds;extension, 72° C., 1 minute, 35 cycles; extension, 72° C. 5 minutes;hold, 4° C., forever. PCR products were run on a 1% agarose gel, stainedwith ethidium bromide, and visualized under UV light using theQuantity-One gel documentation system (Bio-Rad).

Expression of the transgenes was detected in the T1 transgenic line.These results indicated that the transgenes are expressed in thetransgenic lines and strongly suggested that their gene product improvedplant stress tolerance in the transgenic lines. In agreement with theprevious statement, no expression of identical or very similarendogenous genes could be detected by this method. These results are inagreement with the data from Example 7.

Example 10

Engineering Stress-Tolerant Soybeans Plants by Over-Expressing the PK-6,PK-7, PK-8, PK-9, CK-1, CK-2, CK-3, MPK-2, MPK-3, MPK-4, MPK-5, CPK-1and CPK-2 Gene

The constructs pBPSJyw022, pBPSJyw012, pBPSJYW030, PBPSERG010,pBPSSY012, pBPSJyw034, pBPSSY011, pBPSSY016, pBPSJyw014, pBPSJyw025,PBPSERG009, PBPSERG019 and pBPSJyw008 were used to transform soybean asdescribed below.

Seeds of soybean were surface sterilized with 70% ethanol for 4 minutesat room temperature with continuous shaking, followed by 20% (v/v)Clorox supplemented with 0.05% (v/v) Tween for 20 minutes withcontinuous shaking. Then, the seeds were rinsed 4 times with distilledwater and placed on moistened sterile filter paper in a Petri dish atroom temperature for 6 to 39 hours. The seed coats were peeled off, andcotyledons are detached from the embryo axis. The embryo axis wasexamined to make sure that the meristematic region is not damaged. Theexcised embryo axes were collected in a half-open sterile Petri dish andair-dried to a moisture content less than 20% (fresh weight) in a sealedPetri dish until further use.

Agrobacterium tumefaciens culture was prepared from a single colony inLB solid medium plus appropriate antibiotics (e.g. 100 mg/lstreptomycin, 50 mg/l kanamycin) followed by growth of the single colonyin liquid LB medium to an optical density at 600 nm of 0.8. Then, thebacteria culture was pelleted at 7000 rpm for 7 minutes at roomtemperature, and resuspended in MS (Murashige and Skoog, 1962) mediumsupplemented with 100 μM acetosyringone. Bacteria cultures wereincubated in this pre-induction medium for 2 hours at room temperaturebefore use. The axis of soybean zygotic seed embryos at approximately15% moisture content were imbibed for 2 hours at room temperature withthe pre-induced Agrobacterium suspension culture. The embryos areremoved from the imbibition culture and were transferred to Petri dishescontaining solid MS medium supplemented with 2% sucrose and incubatedfor 2 days, in the dark at room temperature. Alternatively, the embryoswere placed on top of moistened (liquid MS medium) sterile filter paperin a Petri dish and incubated under the same conditions described above.After this period, the embryos were transferred to either solid orliquid MS medium supplemented with 500 mg/L carbenicillin or 300 mg/Lcefotaxime to kill the agrobacteria. The liquid medium was used tomoisten the sterile filter paper. The embryos were incubated during 4weeks at 25° C., under 150 μmol m⁻² sec⁻¹ and 12 hours photoperiod. Oncethe seedlings produced roots, they were transferred to sterile metromixsoil. The medium of the iii vitro plants was washed off beforetransferring the plants to soil. The plants were kept under a plasticcover for 1 week to favor the acclimatization process. Then the plantswere transferred to a growth room where they were incubated at 25° C.,under 150 μmol m⁻² sec⁻¹ light intensity and 12 hours photoperiod forabout 80 days.

The transgenic plants were then screened for their improved drought,salt and/or cold tolerance according to the screening method describedin Example 7 demonstrating that transgene expression confers stresstolerance.

Example 11

Engineering Stress-Tolerant Rapeseed/Canola Plants by Over-expressingPKSRP Genes

Canola cotyledonary petioles of 4 day-old young seedlings are used asexplants for tissue culture and transformed according to patentEP1566443. The commercial cultivar Westar (Agriculture Canada) is thestandard variety used for transformation, but other varieties can beused.

Agrobacterium tumefaciens GV3101:pMP90RK containing a binary vector isused for canola transformation. The standard binary vector used fortransformation is pSUN (patent WO02/00900), but many different binaryvector systems have been described for plant transformation (e.g. An, G.in Agrobacterium Protocols. Methods in Molecular Biology vol 44, pp47-62, Gartland KMA and MR Davey eds. Humana Press, Totowa, N.J.). Aplant gene expression cassette consists of at least two genes—aselection marker gene and a plant promoter regulating the transcriptionof the cDNA or genomic DNA of the trait gene. Various selection markergenes can be used including the Arabidopsis gene encoding a mutatedacetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. Nos. 5,767,366 and6,225,105). Similarly, various promoters can be used to regulate thetrait gene to provide constitutive, developmental, tissue orenvironmental regulation of gene transcription. In this example, the 34Spromoter (GenBank Accession numbers M59930 and X16673) is used toprovide constitutive expression of the trait gene.

Canola seeds are surface-sterilized in 70% ethanol for 2 min, incubatedfor 15 min in 55° C. warm tap water and then in 1.5% sodium hypochloritefor 10 min, followed by three rinses with sterilized distilled water.Seeds are then placed on MS medium without hormones, containing GamborgB5 vitamins, 3% sucrose, and 0.8% Oxoidagar. Seeds are germinated at 24°C. for 4 days in low light (<50 μMol/m2s) at 16 hr light. The cotyledonpetiole explants with the cotyledon attached are excised from the invitro seedlings, and inoculated with Agrobacterium by dipping the cutend of the petiole explant into the bacterial suspension. The explantsare then cultured for 3 days on MS medium incl. vitamins containing 3.75mg/l BAP, 3% sucrose, 0.5 g/l MES, pH 5.2, 0.5 mg/l GA3, 0.8% Oxoidagarat 24° C., 16 hr light. After three days of co-cultivation withAgrobacterium, the petiole explants are transferred to regenerationmedium containing 3.75 mg/l BAP, 0.5 mg/l GA3, 0.5 g/l MES, pH 5.2, 300mg/l timentin and selection agent until shoot regeneration. As soon asexplants start to develop shoots, they are transferred to shootelongation medium (A6, containing full strength MS medium includingvitamins, 2% sucrose, 0.5% Oxoidagar, 100 mg/l myo-inositol, 40 mg/ladenine sulfate, 0.5 g/l MES, pH 5.8, 0.0025 mg/l BAP, 0.1 mg/l IBA, 300mg/l timentin and selection agent).

Samples from both in vitro and greenhouse material of the primarytransgenic plants (TO) are analyzed by qPCR using TaqMan probes toconfirm the presence of T-DNA and to determine the number of T-DNAintegrations.

Seed is produced from the primary transgenic plants by self-pollination.The second-generation plants are grown in greenhouse conditions andself-pollinated. The plants are analyzed by qPCR using TaqMan probes toconfirm the presence of T-DNA and to determine the number of T-DNAintegrations. Homozygous transgenic, heterozygous transgenic and azygous(null transgenic) plants are compared for their growth characteristicsand yield.

Example 12

Engineering Stress-Tolerant Corn Plants by Over-Expressing the PKSRPGenes

Agrobacterium cells harboring the genes and the maize ahas gene on thesame plasmid are grown in YP medium supplemented with appropriateantibiotics for 1-3 days. A loop of Agrobacterium cells is collected andsuspended in 2 ml M-LS-002 medium (LS-inf) and the tube containingAgrobactium cells is kept on a shaker for 1-3 hrs at 1,200 rpm.

Corncobs [genotype J553x(HIIIAxA188)] are harvested at 7-12 days afterpollination. The cobs are sterilized in 20% Clorox solution for 15 minfollowed by thorough rinse with sterile water. Immature embryos withsize 0.8-2.0 mm are dissected into the tube containing Agrobacteriumcells in LS-inf solution.

Agro-infection is carried out by keeping the tube horizontally in thelaminar hood at room temperature for 30 min. Mixture of the agroinfection is poured on to a plate containing the co-cultivation medium(M-LS-011). After the liquid agro-solution is piped out, the embryos areplated on the co-cultivation medium with schutellum side up and culturedin the dark at 22° C. for 2-4 days.

Embryos are transferred to M-MS-101 medium without selection. Seven toten days later, embryos are transferred to M-LS-401 medium containing0.75 uM imazethapyr and grown for 4 weeks to select for transformedcallus cells.

Plant regeneration is initiated by transferring resistant calli toM-LS-504 medium supplemented with 0.75 μM imazethapyr and grown underlight at 26° C. for two to three weeks. Regenerated shoots are thentransferred to rooting box with M-MS-607 medium (0.5 μM imazethapyr).

Plantlets with roots are transferred to potting mixture and grown in agrowth chamber for a week, then transplanted to larger pots andmaintained in greenhouse till maturity.

Example 13

Engineering Stress-Tolerant Wheat Plants by Over-Expressing the PK-6,PK-7, PK-8, PK-9, CK-1, CK-2, CK-3, MPK-2, MPK-3, MPK-4, MPK-5, CPK-1and CPK-2

The constructs pBPSJyw022, pBPSJyw012, pBPSJYW030, PBPSERG010,pBPSSY012, pBPSJyw034, pBPSSY011, pBPSSY016, pBPSJyw014, pBPSJyw025,PBPSERG009, PBPSERG019 and pBPSJyw008 were used to transform wheat asdescribed below.

Transformation of wheat is performed with the method described by Ishidaet al. 1996 Nature Biotch. 14745-50. Immature embryos are co-cultivatedwith Agrobacterium tumefaciens that carry “super binary” vectors, andtransgenic plants are recovered through organogenesis. This procedureprovides a transformation efficiency between 2.5% and 20%. Thetransgenic plants are then screened for their improved stress toleranceaccording to the screening method described in Example 7 demonstratingthat transgene expression confers drought tolerance.

Example 14

Greenhouse Screening for Stress Tolerant Plants

High Throughput Drought Performance Screen

Segregating transgenic corn seeds for a transformation event are plantedin small pots. Each of these plants is uniquely labeled, sampled andanalyzed for transgene copy number. Transgene positive and negativeplants are marked and paired with similar sizes for transplantingtogether to large pots. This provides a uniform and competitiveenvironment for the transgene positive and negative plants. The largepots are watered to a certain percentage of the field water capacity ofthe soil depending the severity of water-stress desired. The soil waterlevel is maintained by watering every other day. Plant growth andphysiology traits such as height, stem diameter, leaf rolling, plantwilting, leaf extension rate, leaf water status, chlorophyll content andphotosynthesis rate are measured during the growth period. After aperiod of growth, the above ground portion of the plants is harvested,and the fresh weight and dry weight of each plant are taken. Acomparison of phenotype between the transgene positive and negativeplants is then made.

Water Use Efficiency (WUE) Assay

Transgene positive and negative corn seedlings for a transformationevent are transplanted into a pot with a given amount of soil and water.The pots are covered with caps that permit the seedlings to grow throughbut minimize water loss. Each pot is weighed periodically and wateradded to maintain the initial water content. At the end of theexperiment, the fresh and dry weight of each plant are measured, thewater consumed by each plant is calculated and WUE of each plant iscomputed. Plant growth and physiology traits such as WUE, height, stemdiameter, leaf rolling, plant wilting, leaf extension rate, leaf waterstatus, chlorophyll content and photosynthesis rate are measured duringthe experiment. A comparison of phenotype between the transgene positiveand negative plants is then made.

Desiccation Assay

Segregating transgenic corn seeds for a transformation event are plantedin small pots. These pots are kept in an area in the greenhouse that hasuniform environmental conditions, and cultivated optimally. Each ofthese plants is uniquely labeled, sampled and analyzed for transgenecopy number. The plants are allowed to grow under theses conditionsuntil they reach a predefined growth stage. Water is then withheld.Plant growth and physiology traits such as height, stem diameter, leafrolling, plant wilting, leaf extension rate, leaf water status,chlorophyll content and photosynthesis rate are measured as stressintensity increases. A comparison of the phenotype between transgenepositive and negative plants is then made.

Cycling Drought Assay

Segregating transgenic corn seeds for a transformation event are plantedin small pots. These pots are kept in an area in the greenhouse that hasuniform environmental conditions, and cultivated optimally. Each ofthese plants is uniquely labeled, sampled and analyzed for transgenecopy number. The plants are allowed to grow under theses conditionsuntil they reach a predefined growth stage. Plants are then repeatedlywatered to saturation at a fixed interval of time. This water/droughtcycle is repeated for the duration of the experiment. Plant growth andphysiology traits such as height, stem diameter, leaf rolling, leafextension rate, leaf water status, chlorophyll content andphotosynthesis rate are measured during the growth period. At the end ofthe experiment, the plants are harvested for above-ground fresh and dryweight. A comparison of the phenotype between transgene positive andnegative plants is then made.

Example 15

Field Screening for Stress Tolerant Plants

Segregating Corn Drought-Tolerance Screening Under Rain-Free Conditions

Managed-drought stress at a single location or multiple locations isused. Crop water availability is controlled by drip tape or overheadirrigation at a location which has less than 10 cm rainfall and minimumtemperatures greater than 5° C. expected during an average 5 monthseason, or a location with expected in-season precipitation interceptedby an automated “rain-out shelter” which retracts to provide open fieldconditions when not required. Standard agronomic practices in the areaare followed for soil preparation, planting, fertilization and pestcontrol. Each plot is sown with seed segregating for the presence of asingle transgenic insertion event. A Taqman transgene copy number assayis used on leaf samples to differentiate the transgenics fromnull-segregant control plants. Plants that have been genotyped in thismanner are also scored for a range of phenotypes related todrought-tolerance, growth and yield. These phenotypes include plantheight, grain weight per plant, grain number per plant, ear number perplant, above ground dry-weight, leaf conductance to water vapor, leafCO2 uptake, leaf chlorophyll content, photosynthesis-related chlorophyllfluorescence parameters, water use efficiency, leaf water potential,leaf relative water content, stem sap flow rate, stem hydraulicconductivity, leaf temperature, leaf reflectance, leaf lightabsorptance, leaf area, days to flowering, anthesis-silking interval,duration of grain fill, osmotic potential, osmotic adjustment, rootsize, leaf extension rate, leaf angle, leaf rolling and survival. Allmeasurements are made with commercially available instrumentation forfield physiology, using the standard protocols provided by themanufacturers. Individual plants are used as the replicate unit perevent.

Non-Segregating Corn Drought-Tolerance Screening Under Rain-FreeConditions

Managed-drought stress at a single location or multiple locations isused. Crop water availability is controlled by drip tape or overheadirrigation at a location which has less than 10 cm rainfall and minimumtemperatures greater than 5(C expected during an average 5 month season,or a location with expected in-season precipitation intercepted by anautomated “rain-out shelter” which retracts to provide open fieldconditions when not required. Standard agronomic practices in the areaare followed for soil preparation, planting, fertilization and pestcontrol. Trial layout is designed to pair a plot containing anon-segregating transgenic event with an adjacent plot of null-segregantcontrols. Progeny (or lines derived from the progeny) of a transgenicplant that does not contain the transgene due to Mendelian segregation.Additional replicated paired plots for a particular event aredistributed around the trial. A range of phenotypes related todrought-tolerance, growth and yield are scored in the paired plots andestimated at the plot level. When the measurement technique could onlybe applied to individual plants, these are selected at random each timefrom within the plot. These phenotypes include plant height, grainweight per plant, grain number per plant, ear number per plant, aboveground dry-weight, leaf conductance to water vapor, leaf CO2 uptake,leaf chlorophyll content, photosynthesis-related chlorophyllfluorescence parameters, water use efficiency, leaf water potential,leaf relative water content, stem sap flow rate, stem hydraulicconductivity, leaf temperature, leaf reflectance, leaf lightabsorptance, leaf area, days to flowering, anthesis-silking interval,duration of grain fill, osmotic potential, osmotic adjustment, rootsize, leaf extension rate, leaf angle, leaf rolling and survival. Allmeasurements are made with commercially available instrumentation forfield physiology, using the standard protocols provided by themanufacturers. Individual plots are used as the replicate unit perevent.

Multi-Location Corn Drought-Tolerance and Yield Screening

Five to twenty locations encompassing major corn growing regions areselected. These are widely distributed to provide a range of expectedcrop water availabilities based on average temperature, humidity,precipitation and soil type. Crop water availability is not modifiedbeyond standard agronomic practices. Trial layout is designed to pair aplot containing a non-segregating transgenic event with an adjacent plotof null-segregant controls. A range of phenotypes related todrought-tolerance, growth and yield are scored in the paired plots andestimated at the plot level. When the measurement technique could onlybe applied to individual plants, these are selected at random each timefrom within the plot. These phenotypes included plant height, grainweight per plant, grain number per plant, ear number per plant, aboveground dry-weight, leaf conductance to water vapor, leaf CO2 uptake,leaf chlorophyll content, photosynthesis-related chlorophyllfluorescence parameters, water use efficiency, leaf water potential,leaf relative water content, stem sap flow rate, stem hydraulicconductivity, leaf temperature, leaf reflectance, leaf lightabsorptance, leaf area, days to flowering, anthesis-silking interval,duration of grain fill, osmotic potential, osmotic adjustment, rootsize, leaf extension rate, leaf angle, leaf rolling and survival. Allmeasurements are made with commercially available instrumentation forfield physiology, using the standard protocols provided by themanufacturers. Individual plots are used as the replicate unit perevent.

1. An isolated nucleic acid, wherein the nucleic acid is selected fromthe group consisting of: a) a polynucleotide as defined in SEQ IDNO:129; b) a polynucleotide encoding a polypeptide as defined in SEQ IDNO:130, , SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137,SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ IDNO:142, or SEQ ID NO:143; c) a polynucleotide having at least 70%sequence identity to a polynucleotide as defined in SEQ ID NO:129; andd) a polynucleotide encoding a polypeptide having at least 70% sequenceidentity to a polypeptide as defined in SEQ ID NO:130, , SEQ ID NO:134,SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ i)NO:138, SEQ IDNO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, or SEQ ID NO:143.2. The isolated nucleic acid of claim 1, wherein the nucleic acidcomprises a polynucleotide as defined in SEQ ID NO:129.
 3. The isolatednucleic acid of claim 1, wherein the nucleic acid comprises apolynucleotide having at least 70% sequence identity to a polynucleotideas defined in SEQ ID NO:129.
 4. The isolated nucleic acid of claim 1,wherein the nucleic acid comprises a polynucleotide having at least 80%sequence identity to a polynucleotide as defined in SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:129.
 5. The isolated nucleic acid of claim 1, wherein the nucleicacid comprises a polynucleotide having at least 90% sequence identity toa polynucleotide as defined in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:129.
 6. The isolatednucleic acid of claim 1, wherein the nucleic acid comprises apolynucleotide encoding a polypeptide as defined in SEQ ID NO:130, SEQID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, or SEQ IDNO:143.
 7. The isolated nucleic acid of claim 1, wherein the nucleicacid comprises a polynucleotide encoding a polypeptide having at least70% sequence identity to a polypeptide as defined in SEQ ID NO:130, SEQID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, or SEQ IDNO:143.
 8. The isolated nucleic acid of claim 1, wherein the nucleicacid comprises a polynucleotide encoding a polypeptide having at least80% sequence identity to a polypeptide as defined in SEQ ID NO:130, SEQID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, or SEQ IDNO:143.
 9. The isolated nucleic acid of claim 1, wherein the nucleicacid comprises a polynucleotide encoding a polypeptide having at least90% sequence identity to a polypeptide as defined in SEQ ID NO:130, SEQID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, or SEQ IDNO:143.
 10. The isolated nucleic acid of claim 1, wherein the nucleicacid encodes a polypeptide that functions to increase a plant's growthand/or stress tolerance under normal or stress conditions as compared toa wild type variety of the plant.
 11. A vector comprising the nucleicacid of claim
 1. 12. A transgenic plant cell comprising the nucleic acidof claim
 1. 13. A transgenic plant comprising the plant cell of claim12.
 14. The transgenic plant of claim 13, wherein the plant hasincreased growth and/or increased stress tolerance under normal orstress conditions as compared to a wild type variety of the plant. 15.The transgenic plant of claim 13, wherein the plant is a monocot. 16.The transgenic plant of claim 13, wherein the plant is a dicot.
 17. Thetransgenic plant of claim 13, wherein the plant is selected from thegroup consisting of maize, wheat, rye, oat, triticale, rice, barley,soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower,tagetes, solanaceous plants, potato, tobacco, eggplant, tomato, Viciaspecies, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm,coconut, perennial grass and a forage crop plant.
 18. A plant seedproduced by the plant of claim 13, wherein the seed comprises thenucleic acid.
 19. The seed of claim 18, wherein the seed is truebreeding for an increased growth and/or increased stress tolerance undernormal or stress conditions as compared to a wild type variety of theseed.
 20. A method of producing a transgenic plant containing anisolated nucleic acid, wherein the plant has increased growth and/orincreased stress tolerance under normal or stress conditions as comparedto a wild type variety of the plant comprising, transforming a plantcell with an expression vector comprising the nucleic acid, andgenerating from the plant cell the transgenic plant, wherein the nucleicacid is selected from the group consisting of a) a polynucleotide asdefined in SEQ ID NO:129; b) a polynucleotide encoding a polypeptide asdefined in SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136,SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ IDNO:141, SEQ ID NO:142, or SEQ ID NO:143.; c) a polynucleotide having atleast 70% sequence identity to a polynucleotide as defined in SEQ IDNO:129; and d) a polynucleotide encoding a polypeptide having at least70% sequence identity to a polypeptide as defined in SEQ ID NO:130, SEQID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, or SEQ IDNO:143.
 21. The method of claim 20, wherein the plant is a monocot. 22.The method of claim 20, wherein the plant is a dicot.
 23. The method ofclaim 20, wherein the plant is selected from the group consisting ofmaize, wheat, rye, oat, triticale, rice, barley, soybean, peanut,cotton, rapeseed, canola, manihot, pepper, sunflower, tagetes,solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species,pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut,perennial grass and a forage crop plant.
 24. The method of claim 20,wherein the nucleic acid comprises a polynucleotide as defined inNO:129.
 25. The method of claim 20, wherein the nucleic acid comprises apolynucleotide having at least 70% sequence identity to a polynucleotideas defined in SEQ ID NO:129.
 26. The method of claim 20, wherein thenucleic acid comprises a polynucleotide encoding a polypeptide asdefined in SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136,SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ IDNO:141, SEQ ID NO:142, or SEQ ID NO:143.
 27. The method of claim 20,wherein the nucleic acid comprises a polynucleotide encoding apolypeptide having at least 70% sequence identity to a polypeptide asdefined in SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136,SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ IDNO:141, SEQ ID NO:142, or SEQ ID NO:143.
 28. The method of claim 20,wherein the expression vector comprises a promoter that directsexpression of the nucleic acid.
 29. The method of claim 28, wherein thepromoter is tissue specific.
 30. The method of claim 28, wherein thepromoter is developmentally regulated.